Inhibitors of serine proteases

ABSTRACT

The present invention relates to compounds of formula I: 
                         
or a pharmaceutically acceptable salt or mixtures thereof that inhibit serine protease activity, particularly the activity of hepatitis C virus NS3-NS4A protease.

CROSS-REFERENCE

This application is a continuation of U.S. Ser. No. 11/511,109, filedAug. 28, 2006, and also claims the benefit under 35 U.S.C. §119 of U.S.Application Ser. No. 60/711,530, filed Aug. 26, 2005. Both of theseapplications are incorporated herein in their entirety.

SEQUENCE LISTING

This application incorporates by reference in its entirety the SequenceListing named “seq_list_inhibitors.ST25.txt” created on Aug. 23, 2006,that is 8,192 bytes, and filed electronically herewith on Feb. 28, 2011.

FIELD OF THE INVENTION

The present invention relates to compounds that inhibit serine proteaseactivity, particularly the activity of hepatitis C virus NS3-NS4Aprotease. As such, they act by interfering with the life cycle of thehepatitis C virus and are also useful as antiviral agents. The inventionfurther relates to compositions comprising these compounds either for exvivo use or for administration to a patient suffering from HCVinfection. The invention also relates to methods of treating an HCVinfection in a patient by administering a composition comprising acompound of this invention.

BACKGROUND OF THE INVENTION

Infection by hepatitis C virus (“HCV”) is a compelling human medicalproblem. HCV is recognized as the causative agent for most cases ofnon-A, non-B hepatitis, with an estimated human sero-prevalence of 3%globally [A. Alberti et al., “Natural History of Hepatitis C,” J.Hepatology, 31., (Suppl. 1), pp. 17-24 (1999)]. Nearly four millionindividuals may be infected in the United States alone [M. J. Alter etal., “The Epidemiology of Viral Hepatitis in the United States,Gastroenterol. Clin. North Am., 23, pp. 437-455 (1994); M. J. Alter“Hepatitis C Virus Infection in the United States,” J. Hepatology, 31.,(Suppl. 1), pp. 88-91 (1999)].

Upon first exposure to HCV only about 20% of infected individualsdevelop acute clinical hepatitis while others appear to resolve theinfection spontaneously. In almost 70% of instances, however, the virusestablishes a chronic infection that persists for decades [S. Iwarson,“The Natural Course of Chronic Hepatitis,” FEMS Microbiology Reviews,14, pp. 201-204 (1994); D. Lavanchy, “Global Surveillance and Control ofHepatitis C,” J. Viral Hepatitis, 6, pp. 35-47 (1999)]. This usuallyresults in recurrent and progressively worsening liver inflammation,which often leads to more severe disease states such as cirrhosis andhepatocellular carcinoma [M. C. Kew, “Hepatitis C and HepatocellularCarcinoma”, FEMS Microbiology Reviews, 14, pp. 211-220 (1994); I. Saitoet. al., “Hepatitis C Virus Infection is Associated with the Developmentof Hepatocellular Carcinoma,” Proc. Natl. Acad. Sci. USA, 87, pp.6547-6549 (1990)]. Unfortunately, there are no broadly effectivetreatments for the debilitating progression of chronic HCV.

The HCV genome encodes a polyprotein of 3010-3033 amino acids [Q. L.Choo, et. al., “Genetic Organization and Diversity of the Hepatitis CVirus.” Proc. Natl. Acad. Sci. USA, 88, pp. 2451-2455 (1991); N. Kato etal., “Molecular Cloning of the Human Hepatitis C Virus Genome FromJapanese Patients with Non-A, Non-B Hepatitis,” Proc. Natl. Acad. Sci.USA, 87, pp. 9524-9528 (1990); A. Takamizawa et. al., “Structure andOrganization of the Hepatitis C Virus Genome Isolated From HumanCarriers,” J. Virol., 65, pp. 1105-1113 (1991)]. The HCV nonstructural(NS) proteins are presumed to provide the essential catalytic machineryfor viral replication. The NS proteins are derived by proteolyticcleavage of the polyprotein [R. Bartenschlager et. al., “NonstructuralProtein 3 of the Hepatitis C Virus Encodes a Serine-Type ProteinaseRequired for Cleavage at the NS3/4 and NS4/5 Junctions,” J. Virol., 67,pp. 3835-3844 (1993); A. Grakoui et. al., “Characterization of theHepatitis C Virus-Encoded Serine Proteinase: Determination ofProteinase-Dependent Polyprotein Cleavage Sites,” J. Virol., 67, pp.2832-2843 (1993); A. Grakoui et. al., “Expression and Identification ofHepatitis C Virus Polyprotein Cleavage Products,” J. Virol., 67, pp.1385-1395 (1993); L. Tomei et. al., “NS3 is a serine protease requiredfor processing of hepatitis C virus polyprotein”, J. Virol., 67, pp.4017-4026 (1993)].

The HCV NS protein 3 (NS3) contains a serine protease activity thathelps process the majority of the viral enzymes, and is thus consideredessential for viral replication and infectivity. It is known thatmutations in the yellow fever virus NS3 protease decrease viralinfectivity [Chambers, T. J. et. al., “Evidence that the N-terminalDomain of Nonstructural Protein NS3 From Yellow Fever Virus is a SerineProtease Responsible for Site-Specific Cleavages in the ViralPolyprotein”, Proc. Natl. Acad. Sci. USA, 87, pp. 8898-8902 (1990)]. Thefirst 181 amino acids of NS3 (residues 1027-1207 of the viralpolyprotein) have been shown to contain the serine protease domain ofNS3 that processes all four downstream sites of the HCV polyprotein [C.Lin et al., “Hepatitis C Virus NS3 Serine Proteinase: Trans-CleavageRequirements and Processing Kinetics”, J. Virol., 68, pp. 8147-8157(1994)].

The HCV NS3 serine protease and its associated cofactor, NS4A, helpsprocess all of the viral enzymes, and is thus considered essential forviral replication. This processing appears to be analogous to thatcarried out by the human immunodeficiency virus aspartyl protease, whichis also involved in viral enzyme processing. HIV protease inhibitors,which inhibit viral protein processing, are potent antiviral agents inman indicating that interrupting this stage of the viral life cycleresults in therapeutically active agents. Consequently HCV NS3 serineprotease is also an attractive target for drug discovery.

There are not currently any satisfactory anti-HCV agents or treatments.Until recently, the only established therapy for HCV disease wasinterferon treatment. However, interferons have significant side effects[M. A. Walker et al., “Hepatitis C Virus: An Overview of CurrentApproaches and Progress,” DDT, 4, pp. 518-29 (1999); D. Moradpour etal., “Current and Evolving Therapies for Hepatitis C,” Eur. J.Gastroenterol. Hepatol., 11, pp. 1199-1202 (1999); H. L. A. Janssen etal. “Suicide Associated with Alfa-Interferon Therapy for Chronic ViralHepatitis,” J. Hepatol., 21, pp. 241-243 (1994); P. F. Renault et al.,“Side Effects of Alpha Interferon,” Seminars in Liver Disease, 9, pp.273-277. (1989)] and induce long term remission in only a fraction(˜25%) of cases [O. Weiland, “Interferon Therapy in Chronic Hepatitis CVirus Infection”, FEMS Microbiol. Rev., 14, pp. 279-288 (1994)]. Recentintroductions of the pegylated forms of interferon (PEG-INTRON® andPEGASYS®) and the combination therapy of ribavirin and pegylatedinterferon (REBETROL®) have resulted in only modest improvements inremission rates and only partial reductions in side effects. Moreover,the prospects for effective anti-HCV vaccines remain uncertain.

Thus, there is a need for more effective anti-HCV therapies. Suchinhibitors would have therapeutic potential as protease inhibitors,particularly as serine protease inhibitors, and more particularly as HCVNS3 protease inhibitors. Specifically, such compounds may be useful asantiviral agents, particularly as anti-HCV agents.

SUMMARY OF THE INVENTION

This invention relates to compounds of formula I

or a pharmaceutically acceptable salt thereof wherein,

Each A is —(CX₁X₂)_(a)—;

Each B is —(CX₁X₂)_(b)—;

Each X₁ is independently hydrogen, halo, amino, sulfanyl, optionallysubstituted (C₁₋₄)-aliphatic, optionally substituted aryl, or —O—X_(1A);

Each X₂ is independently hydrogen, halo, amino, sulfanyl, optionallysubstituted (C₁₋₄)-aliphatic, optionally substituted aryl, or —O—X_(1B);

X_(1A) and X_(1B) are each independently an optionally substitutedaliphatic, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl;

Or, X₁ and X₂ together form an oxo group;

Each R₁ is —Z^(A)R₄, wherein each Z^(A) is independently a bond or anoptionally substituted branched or straight C₁₋₁₂ aliphatic chainwherein up to three carbon units of Z^(A) are optionally andindependently replaced by —C(O)—, —C(S)—, —C(O)NR^(A)—,—C(O)NR^(A)NR^(A)—, —C(O)O—, —NR^(A)C(O)O—, —O—, —NR^(A)C(O)NR^(A)—,—NR^(A)NR^(A)—, —S—, —SO—, —SO₂—, —NR^(A)—, —SO₂NR^(A)—, or—NR^(A)SO₂NR^(A)— provided that —NR^(A)NR^(A)—, —NR^(A)C(O)NR^(A)—, or—NR^(A)SO₂NR^(A)— is not directly bound to the nitrogen ring atom offormula I;

Each R₄ is independently R^(A), halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃;

Each R^(A) is independently hydrogen, an optionally substitutedaliphatic, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl;

Each R₂ is —Z^(B)R₅, wherein each Z^(B) is independently a bond or anoptionally substituted branched or straight C₁₋₁₂ aliphatic chainwherein up to three carbon units of Z^(B) are optionally andindependently replaced by —C(O)—, —C(S)—, —C(O)NR^(B)—,—C(O)NR^(B)NR^(B)—, —C(O)O—, —NR^(B)C(O)O—, —NR^(B)C(O)NR^(B)—,—NR^(B)NR^(B)—, —S—, —SO—, —SO₂—, —NR^(B)—, —SO₂NR^(B)—, or—NR^(B)SO₂NR^(B)—, provided that SO, SO₂, or —SO₂NR^(B)— is not directlybound to the carbonyl of formula I;

Each R₅ is independently R^(B), halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃;

Each R^(B) is independently hydrogen, an optionally substitutedaliphatic, an optionally substituted cycloaliphatic, an optionallysubstituted aryl, or an optionally substituted heteroaryl;

Or R₁ and R₂, together with the atoms to which they are attached, forman optionally substituted heterocycloaliphatic ring;

Each R₃ is an optionally substituted aliphatic, amino, sulfonyl,sulfanyl, sulfinyl, sulfonamide, sulfamide, sulfo, —O—R_(3A), anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl;

Each R_(3A) is independently an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl;

Each Y and Y′ is independently —Z^(D)R₇, wherein each Z^(D) isindependently a bond or an optionally substituted straight or branchedC₁₋₆ aliphatic chain wherein up to two carbon units of Z^(D) areoptionally and independently replaced by —C(O)—, —C(S)—, —C(O)NR^(D)—,—C(O)NR^(D)NR^(D)—, —C(O)O—, —NR^(D)C(O)O—, —O—, —NR^(D)C(O)NR^(D)—,—NR^(D)NR^(D)—, —S—, —SO—, —SO₂—, —NR^(D)—, —SO₂NR^(D)—, —NR^(D)SO₂—, or—NR^(D)SO₂NR^(D)—, or Y and Y′ together form ═O or ═S;

Each R₇ is independently R^(D), halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃;

Each R^(D) is independently hydrogen, or optionally substituted aryl;and

Each of a and b is independently 0, 1, 2, or 3; provided that the sum ofa and b is 2 or 3.

In some aspects, the invention features a pharmaceutical compositioncomprising a compound of formula I or a pharmaceutically acceptable saltthereof in an amount effective to inhibit a serine protease; and anacceptable carrier, adjuvant or vehicle. The composition may include anadditional agent selected from an immunomodulatory agent; an antiviralagent; a second inhibitor of HCV protease; an inhibitor of anothertarget in the HCV life cycle; and a cytochrome P-450 inhibitor; orcombinations thereof. The immunomodulatory agent is α-, β-, orγ-interferon or thymosin; said antiviral agent is ribavirin, amantadine,or telbivudine; or said inhibitor of another target in the HCV lifecycle is an inhibitor of HCV helicase, polymerase, or metalloprotease.Cytochrome P-450 inhibitor may be ritonavir.

In other aspects, a method of inhibiting the activity of a serineprotease comprising the step of contacting said serine protease with acompound of formula I. The serine protease may be an HCV NS3 protease.The methods also inluce treating an HCV infection in a patient byadministering a compound of formula I. The method may also includeadministering to said patient an additional agent selected from animmunomodulatory agent; an antiviral agent; a second inhibitor of HCVprotease; an inhibitor of another target in the HCV life cycle; orcombinations thereof; wherein said additional agent is administered tosaid patient in the same dosage form as the serine protease inhibitor oras a separate dosage form. The immunomodulatory agent is α-, β-, orγ-interferon or thymosin; said antiviral agent is ribavarin oramantadine; or said inhibitor of another target in the HCV life cycle isan inhibitor of HCV helicase, polymerase, or metalloprotease.

In still other aspects, a method of eliminating or reducing HCVcontamination of a biological sample or medical or laboratory equipment,includes the step of contacting said biological sample or medical orlaboratory equipment with a compound of formula I. The sample orequipment may be selected from blood, other body fluids, biologicaltissue, a surgical instrument, a surgical garment, a laboratoryinstrument, a laboratory garment, a blood or other body fluid collectionapparatus; a blood or other body fluid storage material.

The compounds of the invention, as described herein, also exhibitadvantageous PK properties and/or increased potency.

The invention also relates to compositions that comprise the abovecompounds and the use thereof; methods of preparing compounds of formulaI, and methods of assaying compounds for serine protease activity. Suchcompositions may be used to pre-treat devices that are to be insertedinto a patient, to treat biological samples, and for directadministration to a patient. In each case, the composition will be usedto lessen the risk of or the severity of the HCV infection.

Definitions

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed. Additionally, generalprinciples of organic chemistry are described in “Organic Chemistry”,Thomas Sorrell, University Science Books, Sausalito: 1999, and “March'sAdvanced Organic Chemistry”, 5^(th) Ed., Ed.: Smith, M. B. and March,J., John Wiley & Sons, New York: 2001, the entire contents of which arehereby incorporated by reference.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the invention.

As used herein the term “aliphatic” encompasses the terms alkyl,alkenyl, alkynyl, each of which being optionally substituted as setforth below.

As used herein, an “alkyl” group refers to a saturated aliphatichydrocarbon group containing 1-8 (e.g., 1-6 or 1-4) carbon atoms. Analkyl group can be straight or branched. Examples of alkyl groupsinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl, or2-ethylhexyl. An alkyl group can be substituted (i.e., optionallysubstituted) with one or more substituents such as halo, phospho,cycloaliphatic [e.g., cycloalkyl or cycloalkenyl], heterocycloaliphatic[e.g., heterocycloalkyl or heterocycloalkenyl], aryl, heteroaryl,alkoxy, aroyl, heteroaroyl, acyl [e.g., (aliphatic)carbonyl,(cycloaliphatic)carbonyl, or (heterocycloaliphatic)carbonyl], nitro,cyano, amido [e.g., (cycloalkylalkyl)carbonylamino, arylcarbonylamino,aralkylcarbonylamino, (heterocycloalkyl)carbonylamino,(heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino,heteroaralkylcarbonylamino alkylaminocarbonyl, cycloalkylaminocarbonyl,heterocycloalkylaminocarbonyl, arylaminocarbonyl, orheteroarylaminocarbonyl], amino [e.g., aliphaticamino,cycloaliphaticamino, or heterocycloaliphaticamino], sulfonyl [e.g.,aliphatic-SO₂—], sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl,sulfamide, oxo, carboxy, carbamoyl, cycloaliphaticoxy,heterocycloaliphaticoxy, aryloxy, heteroaryloxy, aralkyloxy,heteroarylalkoxy, alkoxycarbonyl, alkylcarbonyloxy, or hydroxy. Withoutlimitation, some examples of substituted alkyls include carboxyalkyl(such as HOOC-alkyl, alkoxycarbonylalkyl, and alkylcarbonyloxyalkyl),cyanoalkyl, hydroxyalkyl, alkoxyalkyl, acylalkyl, aralkyl,(alkoxyaryl)alkyl, (sulfonylamino)alkyl (such as(alkyl-SO₂-amino)alkyl), aminoalkyl, amidoalkyl, (cycloaliphatic)alkyl,or haloalkyl.

As used herein, an “alkenyl” group refers to an aliphatic carbon groupthat contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and at least onedouble bond. Like an alkyl group, an alkenyl group can be straight orbranched. Examples of an alkenyl group include, but are not limited to,allyl, isoprenyl, 2-butenyl, and 2-hexenyl. An alkenyl group can beoptionally substituted with one or more substituents such as halo,phospho, cycloaliphatic [e.g., cycloalkyl or cycloalkenyl],heterocycloaliphatic [e.g., heterocycloalkyl or heterocycloalkenyl],aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, acyl [e.g.,(aliphatic)carbonyl, (cycloaliphatic)carbonyl, or(heterocycloaliphatic)carbonyl], nitro, cyano, amido [e.g.,(cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, heteroaralkylcarbonylamino alkylaminocarbonyl,cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl,arylaminocarbonyl, or heteroarylaminocarbonyl], amino [e.g.,aliphaticamino, cycloaliphatic amino, heterocycloaliphaticamino, oraliphaticsulfonylamino], sulfonyl [e.g., alkyl-SO₂—,cycloaliphatic-SO₂—, or aryl-SO₂—], sulfinyl, sulfanyl, sulfoxy, urea,thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl,cycloaliphaticoxy, heterocycloaliphaticoxy, aryloxy, heteroaryloxy,aralkyloxy, heteroaralkoxy, alkoxycarbonyl, alkylcarbonyloxy, orhydroxy. Without limitation, some examples of substituted alkenylsinclude cyanoalkenyl, alkoxyalkenyl, acylalkenyl, hydroxyalkenyl,aralkenyl, (alkoxyaryl)alkenyl, (sulfonylamino)alkenyl (such as(alkyl-SO₂-amino)alkenyl), aminoalkenyl, amidoalkenyl,(cycloaliphatic)alkenyl, or haloalkenyl.

As used herein, an “alkynyl” group refers to an aliphatic carbon groupthat contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and has at least onetriple bond. An alkynyl group can be straight or branched. Examples ofan alkynyl group include, but are not limited to, propargyl and butynyl.An alkynyl group can be optionally substituted with one or moresubstituents such as aroyl, heteroaroyl, alkoxy, cycloalkyloxy,heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, nitro, carboxy,cyano, halo, hydroxy, sulfo, mercapto, sulfanyl [e.g., aliphaticsulfanylor cycloaliphaticsulfanyl], sulfinyl [e.g., aliphaticsulfinyl orcycloaliphaticsulfinyl], sulfonyl [e.g., aliphatic-SO₂—,aliphaticamino-SO₂—, or cycloaliphatic-SO₂—], amido [e.g.,aminocarbonyl, alkylaminocarbonyl, alkylcarbonylamino,cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl,cycloalkylcarbonylamino, arylaminocarbonyl, arylcarbonylamino,aralkylcarbonylamino, (heterocycloalkyl)carbonylamino,(cycloalkylalkyl)carbonylamino, heteroaralkylcarbonylamino,heteroarylcarbonylamino or heteroarylaminocarbonyl], urea, thiourea,sulfamoyl, sulfamide, alkoxycarbonyl, alkylcarbonyloxy, cycloaliphatic,heterocycloaliphatic, aryl, heteroaryl, acyl [e.g.,(cycloaliphatic)carbonyl or (heterocycloaliphatic)carbonyl], amino[e.g., aliphaticamino], sulfoxy, oxo, carboxy, carbamoyl,(cycloaliphatic)oxy, (heterocycloaliphatic)oxy, or (heteroaryl)alkoxy.

As used herein, an “amido” encompasses both “aminocarbonyl” and“carbonylamino”. These terms when used alone or in connection withanother group refers to an amido group such as —N(R^(X))—C(O)—R^(Y) or—C(O)—N(R^(X))₂, when used terminally, and —C(O)—N(R^(X))— or—N(R^(X))—C(O)— when used internally, wherein R^(X) and R^(Y) aredefined below. Examples of amido groups include alkylamido (such asalkylcarbonylamino or alkylaminocarbonyl), (heterocycloaliphatic)amido,(heteroaralkyl)amido, (heteroaryl)amido, (heterocycloalkyl)alkylamido,arylamido, aralkylamido, (cycloalkyl)alkylamido, or cycloalkylamido.

As used herein, an “amino” group refers to —NR^(X)R^(Y) wherein each ofR^(X) and R^(Y) is independently hydrogen, aliphatic, cycloaliphatic,(cycloaliphatic)aliphatic, aryl, araliphatic, heterocycloaliphatic,(heterocycloaliphatic)aliphatic, heteroaryl, carboxy, sulfanyl,sulfinyl, sulfonyl, (aliphatic)carbonyl, (cycloaliphatic)carbonyl,((cycloaliphatic)aliphatic)carbonyl, arylcarbonyl,(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,((heterocycloaliphatic)aliphatic)carbonyl, (heteroaryl)carbonyl, or(heteroaraliphatic)carbonyl, each of which being defined herein andbeing optionally substituted. Examples of amino groups includealkylamino, dialkylamino, or arylamino. When the term “amino” is not theterminal group (e.g., alkylcarbonylamino), it is represented by—NR^(X)—. R^(X) has the same meaning as defined above.

As used herein, an “aryl” group used alone or as part of a larger moietyas in “aralkyl”, “aralkoxy”, or “aryloxyalkyl” refers to monocyclic(e.g., phenyl); bicyclic (e.g., indenyl, naphthalenyl,tetrahydronaphthyl, tetrahydroindenyl); and tricyclic (e.g., fluorenyltetrahydrofluorenyl, or tetrahydroanthracenyl, anthracenyl) ring systemsin which the monocyclic ring system is aromatic or at least one of therings in a bicyclic or tricyclic ring system is aromatic. The bicyclicand tricyclic groups include benzofused 2-3 membered carbocyclic rings.For example, a benzofused group includes phenyl fused with two or moreC₄₋₈ carbocyclic moieties. An aryl is optionally substituted with one ormore substituents including aliphatic [e.g., alkyl, alkenyl, oralkynyl]; cycloaliphatic; (cycloaliphatic)aliphatic;heterocycloaliphatic; (heterocycloaliphatic)aliphatic; aryl; heteroaryl;alkoxy; (cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy;heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy; aroyl;heteroaroyl; amino; oxo (on a non-aromatic carbocyclic ring of abenzofused bicyclic or tricyclic aryl); nitro; carboxy; amido; acyl[e.g., aliphaticcarbonyl; (cycloaliphatic)carbonyl;((cycloaliphatic)aliphatic)carbonyl; (araliphatic)carbonyl;(heterocycloaliphatic)carbonyl;((heterocycloaliphatic)aliphatic)carbonyl; or(heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphatic-SO₂— oramino-SO₂—]; sulfinyl [e.g., aliphatic-S(O)— or cycloaliphatic-S(O)—];sulfanyl [e.g., aliphatic-S—]; cyano; halo; hydroxy; mercapto; sulfoxy;urea; thiourea; sulfamoyl; sulfamide; or carbamoyl. Alternatively, anaryl can be unsubstituted.

Non-limiting examples of substituted aryls include haloaryl [e.g.,mono-, di (such as p,m-dihaloaryl), and (trihalo)aryl]; (carboxy)aryl[e.g., (alkoxycarbonyl)aryl, ((aralkyl)carbonyloxy)aryl, and(alkoxycarbonyl)aryl]; (amido)aryl [e.g., (aminocarbonyl)aryl,(((alkylamino)alkyl)aminocarbonyl)aryl, (alkylcarbonyl)aminoaryl,(arylaminocarbonyl)aryl, and (((heteroaryl)amino)carbonyl)aryl];aminoaryl [e.g., ((alkylsulfonyl)amino)aryl or ((dialkyl)amino)aryl];(cyanoalkyl)aryl; (alkoxy)aryl; (sulfamoyl)aryl [e.g.,(aminosulfonyl)aryl]; (alkylsulfonyl)aryl; (cyano)aryl;(hydroxyalkyl)aryl; ((alkoxy)alkyl)aryl; (hydroxy)aryl,((carboxy)alkyl)aryl; (((dialkyl)amino)alkyl)aryl; (nitroalkyl)aryl;(((alkylsulfonyl)amino)alkyl)aryl; ((heterocycloaliphatic)carbonyl)aryl;((alkylsulfonyl)alkyl)aryl; (cyanoalkyl)aryl; (hydroxyalkyl)aryl;(alkylcarbonyl)aryl; alkylaryl; (trihaloalkyl)aryl;p-amino-m-alkoxycarbonylaryl; p-amino-m-cyanoaryl; p-halo-m-aminoaryl;or (m-(heterocycloaliphatic)-o-(alkyl))aryl.

As used herein, an “araliphatic” such as an “aralkyl” group refers to analiphatic group (e.g., a (C₁₋₄)-alkyl group) that is substituted with anaryl group. “Aliphatic,” “alkyl,” and “aryl” are defined herein. Anexample of an araliphatic such as an aralkyl group is benzyl.

As used herein, an “aralkyl” group refers to an alkyl group (e.g., a(C₁₋₄)-alkyl group) that is substituted with an aryl group. Both “alkyl”and “aryl” have been defined above. An example of an aralkyl group isbenzyl. An aralkyl is optionally substituted with one or moresubstituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl,including carboxyalkyl, hydroxyalkyl, or haloalkyl such astrifluoromethyl], cycloaliphatic [e.g., cycloalkyl or cycloalkenyl],(cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl,heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy,heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, nitro,carboxy, alkoxycarbonyl, alkylcarbonyloxy, amido [e.g., aminocarbonyl,alkylcarbonylamino, cycloalkylcarbonylamino,(cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, or heteroaralkylcarbonylamin], cyano, halo,hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, a “bicyclic ring system” includes 8-12 (e.g., 9, 10, or11) membered structures that form two rings, wherein the two rings haveat least one atom in common (e.g., 2 atoms in common). Bicyclic ringsystems include bicycloaliphatics (e.g., bicycloalkyl orbicycloalkenyl), bicycloheteroaliphatics, bicyclic aryls, and bicyclicheteroaryls.

As used herein, a “cycloaliphatic” group encompasses a “cycloalkyl”group and a “cycloalkenyl” group, each of which being optionallysubstituted as set forth below. As used herein, a “cycloalkyl” grouprefers to a saturated carbocyclic mono- or bicyclic (fused or bridged)ring of 3-10 (e.g., 5-10) carbon atoms. Examples of cycloalkyl groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,adamantyl, norbornyl, cubyl, octahydro-indenyl, decahydro-naphthyl,bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl,bicyclo[3.3.2.]decyl, bicyclo[2.2.2]octyl, adamantyl, azacycloalkyl, or((aminocarbonyl)cycloalkyl)cycloalkyl. A “cycloalkenyl” group, as usedherein, refers to a non-aromatic carbocyclic ring of 3-10 (e.g., 4-8)carbon atoms having one or more double bonds. Examples of cycloalkenylgroups include cyclopentenyl, 1,4-cyclohexa-di-enyl, cycloheptenyl,cyclooctenyl, hexahydro-indenyl, octahydro-naphthyl, cyclohexenyl,cyclopentenyl, bicyclo[2.2.2]octenyl, or bicyclo[3.3.1]nonenyl. Acycloalkyl or cycloalkenyl group can be optionally substituted with oneor more substituents such as phosphor, aliphatic [e.g., alkyl, alkenyl,or alkynyl], cycloaliphatic, (cycloaliphatic) aliphatic,heterocycloaliphatic, (heterocycloaliphatic) aliphatic, aryl,heteroaryl, alkoxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy,aryloxy, heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl,heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino,(cycloaliphatic)carbonylamino, ((cycloaliphatic)aliphatic)carbonylamino,(aryl)carbonylamino, (araliphatic)carbonylamino,(heterocycloaliphatic)carbonylamino,((heterocycloaliphatic)aliphatic)carbonylamino,(heteroaryl)carbonylamino, or (heteroaraliphatic)carbonylamino], nitro,carboxy [e.g., HOOC—, alkoxycarbonyl, or alkylcarbonyloxy], acyl [e.g.,(cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl,(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,((heterocycloaliphatic)aliphatic)carbonyl, or(heteroaraliphatic)carbonyl], cyano, halo, hydroxy, mercapto, sulfonyl[e.g., alkyl-SO₂— and aryl-SO₂—], sulfinyl [e.g., alkyl-S(O)—], sulfanyl[e.g., alkyl-S—], sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, orcarbamoyl.

As used herein, “cyclic moiety” includes cycloaliphatic,heterocycloaliphatic, aryl, or heteroaryl, each of which has beendefined previously.

As used herein, the term “heterocycloaliphatic” encompasses aheterocycloalkyl group and a heterocycloalkenyl group, each of whichbeing optionally substituted as set forth below.

As used herein, a “heterocycloalkyl” group refers to a 3-10 memberedmono- or bicylic (fused or bridged) (e.g., 5- to 10-membered mono- orbicyclic) saturated ring structure, in which one or more of the ringatoms is a heteroatom (e.g., N, O, S, or combinations thereof). Examplesof a heterocycloalkyl group include piperidyl, piperazyl,tetrahydropyranyl, tetrahydrofuryl, 1,4-dioxolanyl, 1,4-dithianyl,1,3-dioxolanyl, oxazolidyl, isoxazolidyl, morpholinyl, thiomorpholyl,octahydrobenzofuryl, octahydrochromenyl, octahydrothiochromenyl,octahydroindolyl, octahydropyrindinyl, decahydroquinolinyl,octahydrobenzo[b]thiopheneyl, 2-oxa-bicyclo[2.2.2]octyl,1-aza-bicyclo[2.2.2]octyl, 3-aza-bicyclo[3.2.1]octyl, and2,6-dioxa-tricyclo[3.3.1.0^(3,7)]nonyl. A monocyclic heterocycloalkylgroup can be fused with a phenyl moiety such as tetrahydroisoquinoline.A “heterocycloalkenyl” group, as used herein, refers to a mono- orbicylic (e.g., 5- to 10-membered mono- or bicyclic) non-aromatic ringstructure having one or more double bonds, and wherein one or more ofthe ring atoms is a heteroatom (e.g., N, O, or S). Monocyclic andbicycloheteroaliphatics are numbered according to standard chemicalnomenclature.

A heterocycloalkyl or heterocycloalkenyl group can be optionallysubstituted with one or more substituents such as phosphor, aliphatic[e.g., alkyl, alkenyl, or alkynyl], cycloaliphatic,(cycloaliphatic)aliphatic, heterocycloaliphatic,(heterocycloaliphatic)aliphatic, aryl, heteroaryl, alkoxy,(cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy, heteroaryloxy,(araliphatic)oxy, (heteroaraliphatic)oxy, aroyl, heteroaroyl, amino,amido [e.g., (aliphatic)carbonylamino, (cycloaliphatic)carbonylamino,((cycloaliphatic) aliphatic)carbonylamino, (aryl)carbonylamino,(araliphatic)carbonylamino, (heterocycloaliphatic)carbonylamino,((heterocycloaliphatic) aliphatic)carbonylamino,(heteroaryl)carbonylamino, or (heteroaraliphatic)carbonylamino], nitro,carboxy [e.g., HOOC—, alkoxycarbonyl, or alkylcarbonyloxy], acyl [e.g.,(cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl,(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,((heterocycloaliphatic)aliphatic)carbonyl, or(heteroaraliphatic)carbonyl], nitro, cyano, halo, hydroxy, mercapto,sulfonyl [e.g., alkylsulfonyl or arylsulfonyl], sulfinyl [e.g.,alkylsulfinyl], sulfanyl [e.g., alkylsulfanyl], sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

A “heteroaryl” group, as used herein, refers to a monocyclic, bicyclic,or tricyclic ring system having 4 to 15 ring atoms wherein one or moreof the ring atoms is a heteroatom (e.g., N, O, S, or combinationsthereof) and in which the monocyclic ring system is aromatic or at leastone of the rings in the bicyclic or tricyclic ring systems is aromatic.A heteroaryl group includes a benzofused ring system having 2 to 3rings. For example, a benzofused group includes benzo fused with one ortwo 4 to 8 membered heterocycloaliphatic moieties (e.g., indolizyl,indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl,benzo[b]thiophenyl, quinolinyl, or isoquinolinyl). Some examples ofheteroaryl are azetidinyl, pyridyl, 1H-indazolyl, furyl, pyrrolyl,thienyl, thiazolyl, oxazolyl, imidazolyl, tetrazolyl, benzofuryl,isoquinolinyl, benzthiazolyl, xanthene, thioxanthene, phenothiazine,dihydroindole, benzo[1,3]dioxole, benzo[b]furyl, benzo[b]thiophenyl,indazolyl, benzimidazolyl, benzthiazolyl, puryl, cinnolyl, quinolyl,quinazolyl,cinnolyl, phthalazyl, quinazolyl, quinoxalyl, isoquinolyl,4H-quinolizyl, benzo-1,2,5-thiadiazolyl, or 1,8-naphthyridyl.

Without limitation, monocyclic heteroaryls include furyl, thiophenyl,2H-pyrrolyl, pyrrolyl, oxazolyl, thazolyl, imidazolyl, pyrazolyl,isoxazolyl, isothiazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl, 4-H-pranyl,pyridyl, pyridazyl, pyrimidyl, pyrazolyl, pyrazyl, or 1,3,5-triazyl.Monocyclic heteroaryls are numbered according to standard chemicalnomenclature.

Without limitation, bicyclic heteroaryls include indolizyl, indolyl,isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl, benzo[b]thiophenyl,quinolinyl, isoquinolinyl, indolizyl, isoindolyl, indolyl,benzo[b]furyl, bexo[b]thiophenyl, indazolyl, benzimidazyl,benzthiazolyl, purinyl, 4H-quinolizyl, quinolyl, isoquinolyl, cinnolyl,phthalazyl, quinazolyl, quinoxalyl, 1,8-naphthyridyl, or pteridyl.Bicyclic heteroaryls are numbered according to standard chemicalnomenclature.

A heteroaryl is optionally substituted with one or more substituentssuch as aliphatic [e.g., alkyl, alkenyl, or alkynyl]; cycloaliphatic;(cycloaliphatic)aliphatic; heterocycloaliphatic;(heterocycloaliphatic)aliphatic; aryl; heteroaryl; alkoxy;(cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy; heteroaryloxy;(araliphatic)oxy; (heteroaraliphatic)oxy; aroyl; heteroaroyl; amino; oxo(on a non-aromatic carbocyclic or heterocyclic ring of a bicyclic ortricyclic heteroaryl); carboxy; amido; acyl [e.g., aliphaticcarbonyl;(cycloaliphatic)carbonyl; ((cycloaliphatic)aliphatic)carbonyl;(araliphatic)carbonyl; (heterocycloaliphatic)carbonyl;((heterocycloaliphatic)aliphatic)carbonyl; or(heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphaticsulfonyl oraminosulfonyl]; sulfinyl [e.g., aliphaticsulfinyl]; sulfanyl [e.g.,aliphaticsulfanyl]; nitro; cyano; halo; hydroxy; mercapto; sulfoxy;urea; thiourea; sulfamoyl; sulfamide; or carbamoyl. Alternatively, aheteroaryl can be unsubstituted.

Non-limiting examples of substituted heteroaryls include(halo)heteroaryl [e.g., mono- and di-(halo)heteroaryl];(carboxy)heteroaryl [e.g., (alkoxycarbonyl)heteroaryl]; cyanoheteroaryl;aminoheteroaryl [e.g., ((alkylsulfonyl)amino)heteroaryl and((dialkyl)amino)heteroaryl]; (amido)heteroaryl [e.g.,aminocarbonylheteroaryl, ((alkylcarbonyl)amino)heteroaryl,((((alkyl)amino)alkyl)aminocarbonyl)heteroaryl,(((heteroaryl)amino)carbonyl)heteroaryl,((heterocycloaliphatic)carbonyl)heteroaryl, and((alkylcarbonyl)amino)heteroaryl]; (cyanoalkyl)heteroaryl;(alkoxy)heteroaryl; (sulfamoyl)heteroaryl [e.g.,(aminosulfonyl)heteroaryl]; (sulfonyl)heteroaryl [e.g.,(alkylsulfonyl)heteroaryl]; (hydroxyalkyl)heteroaryl;(alkoxyalkyl)heteroaryl; (hydroxy)heteroaryl;((carboxy)alkyl)heteroaryl; (((dialkyl)amino)alkyl]heteroaryl;(heterocycloaliphatic)heteroaryl; (cycloaliphatic)heteroaryl;(nitroalkyl)heteroaryl; (((alkylsulfonyl)amino)alkyl)heteroaryl;((alkylsulfonyl)alkyl)heteroaryl; (cyanoalkyl)heteroaryl;(acyl)heteroaryl [e.g., (alkylcarbonyl)heteroaryl]; (alkyl)heteroaryl,and (haloalkyl)heteroaryl [e.g., trihaloalkylheteroaryl].

A “heteroaraliphatic (such as a heteroaralkyl group) as used herein,refers to an aliphatic group (e.g., a (C₁₋₄)-alkyl group) that issubstituted with a heteroaryl group. “Aliphatic,” “alkyl,” and“heteroaryl” have been defined above.

A “heteroaralkyl” group, as used herein, refers to an alkyl group (e.g.,a (C₁₋₄)-alkyl group) that is substituted with a heteroaryl group. Both“alkyl” and “heteroaryl” have been defined above. A heteroaralkyl isoptionally substituted with one or more substituents such as alkyl(including carboxyalkyl, hydroxyalkyl, and haloalkyl such astrifluoromethyl), alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl,heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy,cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy,heteroaralkyloxy, aroyl, heteroaroyl, nitro, carboxy, alkoxycarbonyl,alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino,cycloalkylcarbonylamino, (cycloalkylalkyl)carbonylamino,arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo,hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, an “acyl” group refers to a formyl group or R^(X)—C(O)—(such as alkyl-C(O)—, also referred to as “alkylcarbonyl”) where R^(X)and “alkyl” have been defined previously. Acetyl and pivaloyl areexamples of acyl groups.

As used herein, an “aroyl” or “heteroaroyl” refers to an aryl-C(O)— or aheteroaryl-C(O)—. The aryl and heteroaryl portion of the aroyl orheteroaroyl is optionally substituted as previously defined.

As used herein, an “alkoxy” group refers to an alkyl-O— group where“alkyl” has been defined previously.

As used herein, a “carbamoyl” group refers to a group having thestructure —O—CO—NR^(X)R^(Y) or —NR^(X)—CO—O—R^(Z) wherein R^(X) andR^(Y) have been defined above and R^(Z) can be aliphatic, aryl,araliphatic, heterocycloaliphatic, heteroaryl, or heteroaraliphatic.

As used herein, a “carboxy” group refers to —COOH, —COOR^(X), —OC(O)H,—OC(O)R^(X) when used as a terminal group; or —OC(O)— or —C(O)O— whenused as an internal group.

As used herein, a “haloaliphatic” group refers to an aliphatic groupsubstituted with 1-3 halogen. For instance, the term haloalkyl includesthe group —CF₃.

As used herein, a “mercapto” group refers to —SH.

As used herein, a “sulfo” group refers to —SO₃H or —SO₃R^(X) when usedterminally or —S(O)₃— when used internally.

As used herein, a “sulfamide” group refers to the structure—NR^(X)—S(O)₂—NR^(Y)R^(Z) when used terminally and —NR^(X)—S(O)₂—NR^(Y)—when used internally, wherein R^(X), R^(Y), and R^(Z) have been definedabove.

As used herein, a “sulfonamide” group refers to the structure—S(O)₂—NR^(X)R^(Y) or —NR^(X)—S(O)₂—R^(Z) when used terminally; or—S(O)₂—NR^(X)— or —NR^(X)—S(O)₂— when used internally, wherein R^(X),R^(Y), and R^(Z) are defined above.

As used herein a “sulfanyl” group refers to —S—R^(X) when usedterminally and —S— when used internally, wherein R^(X) has been definedabove. Examples of sulfanyls include aliphatic-S—, cycloaliphatic-S—,aryl-S—, or the like.

As used herein a “sulfinyl” group refers to —S(O)—R^(X) when usedterminally and —S(O)— when used internally, wherein R^(X) has beendefined above. Exemplary sulfinyl groups include aliphatic-S(O)—,aryl-S(O)—, (cycloaliphatic(aliphatic)) —S(O)—, cycloalkyl-S(O)—,heterocycloaliphatic-S(O)—, heteroaryl-S(O)—, or the like.

As used herein, a “sulfonyl” group refers to —S(O)₂—R^(X) when usedterminally and —S(O)₂— when used internally, wherein R^(X) has beendefined above. Exemplary sulfonyl groups include aliphatic-S(O)₂—,aryl-S(O)₂—, (cycloaliphatic(aliphatic))-S(O)₂—, cycloaliphatic-S(O)₂—,heterocycloaliphatic-S(O)₂—, heteroaryl-S(O)₂—,(cycloaliphatic(amido(aliphatic)))-S(O)₂— or the like.

As used herein, a “sulfoxy” group refers to —O—SO—R^(X) or —SO—O—R^(X),when used terminally and —O—S(O)— or —S(O)—O— when used internally,where R^(X) has been defined above. As used herein, a “halogen” or“halo” group refers to fluorine, chlorine, bromine or iodine. As usedherein, an “alkoxycarbonyl,” which is encompassed by the term carboxy,used alone or in connection with another group refers to a group such asalkyl-O—C(O)—. As used herein, an “alkoxyalkyl” refers to an alkyl groupsuch as alkyl-O-alkyl-, wherein alkyl has been defined above.

As used herein, a “carbonyl” refer to —C(O)—.

As used herein, an “oxo” refers to ═O.

As used herein, the term “phospho” refers to phosphinates andphosphonates. Examples of phosphinates and phosphonates include—P(O)(R^(P))₂, wherein R^(P) is aliphatic, alkoxy, aryloxy,heteroaryloxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy aryl,heteroaryl, cycloaliphatic or amino.

As used herein, an “aminoalkyl” refers to the structure(R^(X))₂N-alkyl-.

As used herein, a “cyanoalkyl” refers to the structure (NC)-alkyl-.

As used herein, a “urea” group refers to the structure—NR^(X)—CO—NR^(Y)R^(Z) and a “thiourea” group refers to the structure—NR^(X)—CS—NR^(Y)R^(Z) when used terminally and —NR^(X)—CO—NR^(Y)— or—NR^(X)—CS—NR^(Y)— when used internally, wherein R^(X), R^(Y), and R^(Z)have been defined above.

As used herein, a “guanidine” group refers to the structure—N═C(N(R^(X)R^(Y))N(R^(X)R^(Y)) or —NR^(X)—C(═NR^(X))NR^(X)R^(Y) whereinR^(X) and R^(Y) have been defined above.

As used herein, the term “amidino” group refers to the structure—C═(NR^(X))N(R^(X)R^(Y)) wherein R^(X) and R^(Y) have been definedabove.

In general, the term “vicinal” refers to the placement of substituentson a group that includes two or more carbon atoms, wherein thesubstituents are attached to adjacent carbon atoms.

In general, the term “geminal” refers to the placement of substituentson a group that includes two or more carbon atoms, wherein thesubstituents are attached to the same carbon atom.

The terms “terminally” and “internally” refer to the location of a groupwithin a substituent. A group is terminal when the group is present atthe end of the substituent not further bonded to the rest of thechemical structure. Carboxyalkyl, i.e., R^(X)O(O)C-alkyl is an exampleof a carboxy group used terminally. A group is internal when the groupis present in the middle of a substituent of the chemical structure.Alkylcarboxy (e.g., alkyl-C(O)O— or alkyl—OC(O)—) and alkylcarboxyaryl(e.g., alkyl-C(O)O-aryl- or alkyl-O(CO)-aryl-) are examples of carboxygroups used internally.

As used herein, “cyclic group” includes mono-, bi-, and tri-cyclic ringsystems including cycloaliphatic, heterocycloaliphatic, aryl, orheteroaryl, each of which has been previously defined.

As used herein, a “bridged bicyclic ring system” refers to a bicyclicheterocyclicalipahtic ring system or bicyclic cycloaliphatic ring systemin which the rings are bridged. Examples of bridged bicyclic ringsystems include, but are not limited to, adamantanyl, norbornanyl,bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl,bicyclo[3.2.3]nonyl, 2-oxabicyclo[2.2.2]octyl, 1-azabicyclo[2.2.2]octyl,3-azabicyclo[3.2.1]octyl, and 2,6-dioxa-tricyclo[3.3.1.0^(3,7)]nonyl. Abridged bicyclic ring system can be optionally substituted with one ormore substituents such as alkyl (including carboxyalkyl, hydroxyalkyl,and haloalkyl such as trifluoromethyl), alkenyl, alkynyl, cycloalkyl,(cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl,heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy,heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, nitro,carboxy, alkoxycarbonyl, alkylcarbonyloxy, aminocarbonyl,alkylcarbonylamino, cycloalkylcarbonylamino,(cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo,hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, an “aliphatic chain” refers to a branched or straightaliphatic group (e.g., alkyl groups, alkenyl groups, or alkynyl groups).A straight aliphatic chain has the structure —[CH₂]_(v)—, where v is1-6. A branched aliphatic chain is a straight aliphatic chain that issubstituted with one or more aliphatic groups. A branched aliphaticchain has the structure —[CHQ]_(v)- where Q is hydrogen or an aliphaticgroup; however, Q shall be an aliphatic group in at least one instance.The term aliphatic chain includes alkyl chains, alkenyl chains, andalkynyl chains, where alkyl, alkenyl, and alkynyl are defined above.

The phrase “optionally substituted” is used interchangeably with thephrase “substituted or unsubstituted.” As described herein, compounds ofthe invention can optionally be substituted with one or moresubstituents, such as are illustrated generally above, or as exemplifiedby particular classes, subclasses, and species of the invention. Asdescribed herein, the variables R₁, R₂, and R₃, and other variablescontained in formulae described herein encompass specific groups, suchas alkyl and aryl. Unless otherwise noted, each of the specific groupsfor the variables R₁, R₂, and R₃, and other variables contained thereincan be optionally substituted with one or more substituents describedherein. Each substituent of a specific group is further optionallysubstituted with one to three of halo, cyano, oxo, alkoxy, hydroxy,amino, nitro, aryl, cycloaliphatic, heterocycloaliphatic, heteroaryl,haloalkyl, and alkyl. For instance, an alkyl group can be substitutedwith alkylsulfanyl and the alkylsulfanyl can be optionally substitutedwith one to three of halo, cyano, oxo, alkoxy, hydroxy, amino, nitro,aryl, haloalkyl, and alkyl. As an additional example, the cycloalkylportion of a (cycloalkyl)carbonylamino can be optionally substitutedwith one to three of halo, cyano, alkoxy, hydroxy, nitro, haloalkyl, andalkyl. When two alkoxy groups are bound to the same atom or adjacentatoms, the two alkxoy groups can form a ring together with the atom(s)to which they are bound.

In general, the term “substituted,” whether preceded by the term“optionally” or not, refers to the replacement of hydrogen radicals in agiven structure with the radical of a specified substituent. Specificsubstituents are described above in the definitions and below in thedescription of compounds and examples thereof. Unless otherwiseindicated, an optionally substituted group can have a substituent ateach substitutable position of the group, and when more than oneposition in any given structure can be substituted with more than onesubstituent selected from a specified group, the substituent can beeither the same or different at every position. A ring substituent, suchas a heterocycloalkyl, can be bound to another ring, such as acycloalkyl, to form a spiro-bicyclic ring system, e.g., both rings shareone common atom. As one of ordinary skill in the art will recognize,combinations of substituents envisioned by this invention are thosecombinations that result in the formation of stable or chemicallyfeasible compounds.

The phrase “stable or chemically feasible,” as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and preferablytheir recovery, purification, and use for one or more of the purposesdisclosed herein. In some embodiments, a stable compound or chemicallyfeasible compound is one that is not substantially altered when kept ata temperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week.

As used herein, an effective amount is defined as the amount required toconfer a therapeutic effect on the treated patient, and is typicallydetermined based on age, surface area, weight, and condition of thepatient. The interrelationship of dosages for animals and humans (basedon milligrams per meter squared of body surface) is described byFreireich et al., Cancer Chemother. Rep., 50: 219 (1966). Body surfacearea may be approximately determined from height and weight of thepatient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley,N.Y., 537 (1970). As used herein, “patient” refers to a mammal,including a human.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention. Unless otherwise stated, alltautomeric forms of the compounds of the invention are within the scopeof the invention. Additionally, unless otherwise stated, structuresdepicted herein are also meant to include compounds that differ only inthe presence of one or more isotopically enriched atoms. For example,compounds having the present structures except for the replacement ofhydrogen by deuterium or tritium, or the replacement of a carbon by a¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Suchcompounds are useful, for example, as analytical tools or probes inbiological assays, or as therapeutic agents.

In other aspects, the invention features certain compounds as describedgenerically and specifically below. Such specific descriptions areillustrative only and are not meant to limit scope of the compounds oruses thereof.

I. COMPOUNDS

A. Generic Compounds

In some aspects, the invention provides compounds of formula I usefulfor inhibiting serine protease activity and methods of inhibiting serineprotease activity. Compounds of formula I include:

or a pharmaceutically acceptable salt thereof wherein,

Each A is —(CX₁X₂)_(a)—;

Each B is —(CX₁X₂)_(b)—;

Each X₁ is independently hydrogen, halo, amino, sulfanyl, optionallysubstituted (C₁₋₄)-aliphatic, optionally substituted aryl, or —O—X_(1A);

Each X₂ is independently hydrogen, halo, amino, sulfanyl, optionallysubstituted (C₁₋₄)-aliphatic, optionally substituted aryl, or —O—X_(1B);

X_(1A) and X_(1B) are each independently an optionally substitutedaliphatic, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl;

Or, X₁ and X₂ together form an oxo group;

Each R₁ is —Z^(A)R₄, wherein each Z^(A) is independently a bond or anoptionally substituted branched or straight C₁₋₁₂ aliphatic chainwherein up to three carbon units of Z^(A) are optionally andindependently replaced by —C(O)—, —C(S)—, —C(O)NR^(A)—,—C(O)NR^(A)NR^(A)—, —C(O)O—, —NR^(A)C(O)O—, —O—, —NR^(A)C(O)NR^(A)—,—NR^(A)NR^(A)—, —S—, —SO—, —SO₂—, —NR^(A)—, —SO₂NR^(A)—, or—NR^(A)SO₂NR^(A)— provided that —NR^(A)NR^(A)—, —NR^(A)C(O)NR^(A)—, or—NR^(A)SO₂NR^(A)— is not directly bound to the nitrogen ring atom offormula I;

Each R₄ is independently R^(A), halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃;

Each R^(A) is independently hydrogen, an optionally substitutedaliphatic, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl;

Each R₂ is —Z^(B)R₅, wherein each Z^(B) is independently a bond or anoptionally substituted branched or straight C₁₋₁₂ aliphatic chainwherein up to three carbon units of Z^(B) are optionally andindependently replaced by —C(O)—, —C(S)—, —C(O)NR^(B)—,—C(O)NR^(B)NR^(B)—, —C(O)O—, —NR^(B)C(O)O—, —NR^(B)C(O)NR^(B)—,—NR^(B)NR^(B)—, —S—, —SO—, —SO₂—, —NR^(B)—, —SO₂NR^(B)—, or—NR^(B)SO₂NR^(B)—, provided that SO, SO₂, or —SO₂NR^(B)— is not directlybound to the carbonyl of formula I;

Each R₅ is independently R^(B), halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃;

Each R^(B) is independently hydrogen, an optionally substitutedaliphatic, an optionally substituted cycloaliphatic, an optionallysubstituted aryl, or an optionally substituted heteroaryl;

Or R₁ and R₂, together with the atoms to which they are attached, forman optionally substituted heterocycloaliphatic ring;

Each R₃ is an optionally substituted aliphatic, amino, sulfonyl,sulfanyl, sulfinyl, sulfonamide, sulfamide, sulfo, —O—R_(3A), anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl;

Each R_(3A) is independently an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl;

Each Y and Y′ is independently —Z^(D)R₇, wherein each Z^(D) isindependently a bond or an optionally substituted straight or branchedC₁₋₆ aliphatic chain wherein up to two carbon units of Z^(D) areoptionally and independently replaced by —C(O)—, —C(S)—, —C(O)NR^(D)—,—C(O)NR^(D)NR^(D)—, —C(O)O—, —NR^(D)C(O)O—, —O—, —NR^(D)C(O)NR^(D)—,—NR^(D)NR^(D)—, —S—, —SO—, —SO₂—, —NR^(D)—, —SO₂NR^(D)—, —NR^(D)SO₂—, or—NR^(D)SO₂NR^(D)—, or Y and Y′ together form ═O or ═S;

Each R₇ is independently R^(D), halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃;

Each R^(D) is independently hydrogen, or optionally substituted aryl;and

Each of a and b is independently 0, 1, 2, or 3; provided that the sum ofa and b is 2 or 3.

B. Specific Compounds

1. Substituent R₁:

Each R₁ is —Z^(A)R₄, wherein each Z^(A) is independently a bond or anoptionally substituted branched or straight C₁₋₁₂ aliphatic chainwherein up to three carbon units of Z^(A) are optionally andindependently replaced by —C(O)—, —C(S)—, —C(O)NR^(A)—,—C(O)NR^(A)NR^(A)—, —C(O)O—, —NR^(A)C(O)O—, —O—, —NR^(A)C(O)NR^(A)—,—NR^(A)NR^(A)—, —S—, —SO—, —SO₂—, —NR^(A)—, —SO₂NR^(A)—, or—NR^(A)SO₂NR^(A)— provided that —NR^(A)NR^(A)—, —NR^(A)C(O)NR^(A)—, or—NR^(A)SO₂NR^(A)— is not directly bound to the nitrogen ring atom offormula I.

Each R₄ is independently R^(A), halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃.

Each R^(A) is independently hydrogen, an optionally substitutedaliphatic, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl.

In several embodiments R₁ is optionally substituted with 1 to 4substituents.

In certain embodiments, R₁ is -Q₄-W₄-Q₃-W₃-Q₂-W₂-Q₁; wherein each of W₂,W₃, and W₄ is independently a bond, —C(O)—, —C(S)—, —C(O)N(Q₅)—,—C(O)O—, —O—, —N(Q₅)C(O)N(Q₅)-, —SO₂—, —N(Q₅)SO₂—, —S—, —N(Q₅)-, —SO—,—OC(O)—, —N(Q₅)C(O)O—, or —SO₂N(Q₅)—; each of Q₁, Q₂, Q₃ and Q₄ isindependently a bond, an optionally substituted C₁₋₄ aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, an optionallysubstituted heteroaryl, or a hydrogen when Q₁, Q₂, Q₃, or Q₄ is theterminal group of R₁; and each Q₅ is independently hydrogen or anoptionally substituted aliphatic. In some specific embodiments, Q₄ is abond.

In several embodiments, R₁ is an optionally substituted acyl group. Inseveral examples, R₁ is an optionally substituted alkylcarbonyl.Additional examples of R₁ include (amino)alkylcarbonyl,(halo)alkylcarbonyl, (aryl)alkylcarbonyl, (cycloaliphatic)alkylcarbonyl,or (heterocycloaliphatic)alkylcarbonyl. Included in these examples areembodiments where R₁ is(heterocycloalkyl(oxy(carbonyl(amino))))alkylcarbonyl,(heteroaryl(carbonyl(amino(alkyl(carbonyl(amino)))))alkylcarbonyl,(bicycloaryl(sulfonyl(amino)))alkylcarbonyl,(aryl(alkoxy(carbonyl(amino))))alkylcarbonyl,(alkyl(carbonyl(amino)))alkylcarbonyl,(alkenyl(alkoxy(carbonyl(amino))))alkylcarbonyl,(cycloaliphatic(alkyl(amino(carbonyl(amino)))))alkylcarbonyl,(heteroaryl(carbonyl(amino(alkyl(carbonyl(amino))))))alkylcarbonyl,(alkyl(amino(carbonyl(amino))))alkylcarbonyl, or(bicycloaryl(amino(carbonyl(amino))))alkylcarbonyl, each of which isoptionally substituted with 1-3 substituents.

In several embodiments, R₁ is an optionally substituted carboxy group.In one example, R₁ is optionally substituted alkoxycarbonyl. Anotherexample of R₁ includes (C₁₋₄)-alkoxycarbonyl, or (tricyclicaryl)alkoxycarbonyl, each of which is optionally substituted with 1-3substituents. Other carboxy groups include (aliphatic(oxy))carbonyl, a(heteroaralkyl(oxy))carbonyl, (heterocycloaliphatic(oxy)carbonyl,(aralkyl(oxy))carbonyl, each of which is optionally substituted with 1-3of halo, alkoxy, aliphatic, cycloaliphatic, heterocycloaliphatic, aryl,heteroaryl, or combinations thereof.

In several embodiments, R₁ is optionally substituted aminocarbonyl.Examples of R₁ include (alkoxy(aryl(alkyl)))aminocarbonyl,(alkyl)aminocarbonyl, or(aryl(alkoxy(carbonyl(alkyl(amino(carbonyl(alkyl)))))))aminocarbonyl,each of which is optionally substituted with 1-3 substituents.

In several embodiments, R₁ is optionally substituted heteroaryl. In oneexample, R₁ is an optionally substituted oxazolyl, pyrrolyl, furyl,thiophenyl, triazinyl, pyridinyl, pyrazinyl, pyrimidinyl, orpyridazinyl.

In several embodiments, R₁ is an alkylsulfonyl, aminosulfonyl,arylsulfonyl, heteroarylsulfonyl, cycloaliphaticsulfonyl, orheterocycloaliphaticsulfonyl, each of which is optionally substitutedwith 1-4 substituents.

In several embodiments, R₁ is an optionally substituted alkylsulfonyl.Examples of R₁ include (aryl)alkylsulfonyl, or(alkyl(amino))alkylsulfonyl, each of which is optionally substitutedwith 1-3 substituents. alkylsulfonyl, aminosulfonyl, arylsulfonyl,heteroarylsulfonyl, cycloaliphaticsulfonyl, orheterocycloaliphaticsulfonyl, each of which is optionally substituted.In certain embodiments, R₁ is an optionally substituted alkylsulfonyl.

The compound of claim 11, wherein R₁ is (aryl)alkylsulfonyl, or(alkyl(amino))alkylsulfonyl, each of which is optionally substituted.

In some specific embodiments, R₁ is (amino)alkylcarbonyl,(halo)alkylcarbonyl, (aryl)alkylcarbonyl, (cycloaliphatic)alkylcarbonyl,or (heterocycloaliphatic)alkylcarbonyl,(heterocycloalkyl(oxy(carbonyl(amino))))alkylcarbonyl,(heteroaryl(carbonyl(amino(alkyl(carbonyl(amino)))))alkylcarbonyl,(bicycloaryl(sulfonyl(amino)))alkylcarbonyl,(aryl(alkoxy(carbonyl(amino))))alkylcarbonyl,(alkyl(carbonyl(amino)))alkylcarbonyl,(alkenyl(alkoxy(carbonyl(amino))))alkylcarbonyl,(cycloaliphatic(alkyl(amino(carbonyl(amino)))))alkylcarbonyl,(heteroaryl(carbonyl(amino(alkyl(carbonyl(amino))))))alkylcarbonyl,(alkyl(amino(carbonyl(amino))))alkylcarbonyl, or(bicycloaryl(amino(carbonyl(amino))))alkylcarbonyl, each of which isoptionally substituted.

In other specific embodiments, R₁ is a heteroarylcarbonyl, a(cycloaliphatic(alkyl(amido(alkyl))))carbonyl, a(heterocycloaliphatic(oxy(amido(alkyl))))carbonyl, an(aryl(sulfonyl(amino(alkyl))))carbonyl, an(aralkyl(oxy(amido(alkyl))))carbonyl, an(aliphatic(oxy(amido(alkyl))))carbonyl, a(cycloaliphatic(alkyl(amido(alkyl))))carbonyl, a(heterocycloaliphatic)carbonyl, or a(heteroaryl(amido(alkyl(amido(alkyl))))carbonyl, each of which isoptionally substituted with 1-4 of halo, aliphatic, cycloaliphatic,acyl, alkoxy, or combinations thereof.

In still other embodiments, R₁ is amido. For example, R₁ is(alkoxy(aryl(alkyl)))aminocarbonyl, (alkyl)aminocarbonyl, or(aryl(alkoxy(carbonyl(alkyl(amino(carbonyl(alkyl)))))))aminocarbonyl,each of which is optionally substituted.

In several embodiments, R₁ is

wherein T is a bond, —C(O)—, —OC(O)—, —NHC(O)—, —S(O)₂N(H)—, —C(O)C(O)—or —SO₂—; each R is independently hydrogen, amino, an optionallysubstituted aliphatic, an optionally substituted cycloaliphatic, anoptionally substituted heterocycloaliphatic, an optionally substitutedaryl, or an optionally substituted heteroaryl; each R₈ and R′₈ isindependently hydrogen, an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl; and each R₉ is independently hydrogen, anoptionally substituted aliphatic, an optionally substituted heteroaryl,an optionally substituted phenyl, or R₈ and R₉, bound on adjacent atoms,taken together with the atoms to which they are attached form a 5 to 7membered, optionally substituted monocyclic heterocycloaliphatic, or a 6to 12 membered, optionally substituted bicyclic heterocycloaliphatic; orR₈ and R′₈, taken together with the atoms to which they are attachedform an optionally substituted cycloaliphatic or an optionallysubstituted heterocycloaliphatic. For clarity, when R₁ is QVI, each ofR₈, R′₈ and R₉ in each subunit can be independently selected asdescribed above. The set of R₈, R′₈ and R₉ variables in one subunit neednot necessarily be identical to the same set of R₈, R′₈ and R₉ variablesin the other subunit.

In other embodiments, R₁ is QI or QII.

In some embodiments, R in the substituent in QI, QII, QIII, QIV, QV, orQVI is

In other embodiments, R₁ is QVI and R is

In other embodiments, R in the substituent in QI, QII, QIII, QIV, QV, orQVI is

wherein each R₁₀ and R′₁₀ is independently hydrogen, optionallysubstituted aliphatic, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocycloaliphatic, oroptionally substituted cycloaliphatic, or R₁₀ and R′₁₀ together with theatom to which they are both bound form an optionally substitutedcycloaliphatic or an optionally substituted heterocycloaliphatic; andeach K is independently a bond, (C₁₋₁₂)-aliphatic, —O—, —S—, —S(O)₂—,—NR₁₄—, —C(O)—, or —C(O)NR₁₄—, wherein R₁₄ is hydrogen or an optionallysubstituted (C₁₋₁₂)-aliphatic; and n is 1-3. For clarity, when more thanone R₁₀ is present in QI, QII, QIII, QIV, QV, or QVI, each R₁₀ can bethe same or different. In several embodiments, R₁₀ or R′₁₀ is[(C₃₋₁₀)-cycloalkyl or cycloalkenyl]-(C₁₋₁₂)-aliphatic, (3 to 10membered)-heterocycloaliphatic, (3 to 10membered)-heterocycloaliphatic-(C₁₋₁₂)-aliphatic-, (5 to 10membered)-heteroaryl, or (5 to 10membered)-heteroaryl-(C₁₋₁₂)-aliphatic-.

In still other embodiments, R in the substituent in QI, QII, QIII, QIV,QV, or QVI is

In further embodiments, R in the substituent in QI, QII, QIII, QIV, QV,or QVI is

wherein each Z is independently —O—, —S—, —NR₅₀—, or —C(R₅₀)₂—,

is independently a single bond or a double bond, and each R₅₀ isindependently hydrogen, optionally substituted aliphatic, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted heterocycloaliphatic, or optionally substitutedcycloaliphatic; and n is 1 or 2.

In several embodiments, R₁ is

wherein wherein T is a bond, —C(O)—, —OC(O)—, —NHC(O)—, —S(O)₂N(H)—,—C(O)C(O)— or —SO₂—; each R is independently hydrogen, amino, anoptionally substituted aliphatic, an optionally substitutedcycloaliphatic, an optionally substituted heterocycloaliphatic, anoptionally substituted aryl, or an optionally substituted heteroaryl;each R₈ and R′₈ is independently hydrogen, an optionally substitutedaliphatic, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl; and each R₉ is independentlyhydrogen, an optionally substituted aliphatic, an optionally substitutedheteroaryl, an optionally substituted phenyl, or R₈ and R₉, bound onadjacent atoms, taken together with the atoms to which they are attachedform a 5 to 7 membered, optionally substituted monocyclicheterocycloaliphatic, or a 6 to 12 membered, optionally substitutedbicyclic heterocycloaliphatic, in which each heterocycloaliphatic ring;or R₈ and R′₈, taken together with the atoms to which they are attachedform an optionally substituted cycloaliphatic or an optionallysubstituted heterocycloaliphatic; each R₁₁ and R′₁₁ is independentlyhydrogen, an optionally substituted aliphatic, an optionally substitutedheteroaryl, an optionally substituted phenyl, an optionally substitutedcycloaliphatic, an optionally substituted heterocycloaliphatic; or R₁₁and R′₁₁ together with the atom to which they are both attached form anoptionally substituted 3 to 7 membered cycloaliphatic orheterocycloaliphatic ring; and each R₁₂ is independently hydrogen or aprotecting group.

In some embodiments, R₁₁ and R′₁₁ together with the atom to which theyare attached form a 3 to 7 membered ring. Non-limiting examples include:

Non-limiting examples of R₈ and R₁₁ include:

Alternatively, R₈ and R₁₁ together with the atoms to which they areattached may form an optionally substituted 5 to 7 membered monocyclicheterocycloaliphatic or an optionally substituted 6 to 12 memberedbicyclic heterocycloaliphatic, in which each heterocycloaliphatic oraryl ring optionally contains an additional heteroatom selected from O,S and N.

Also, R₈ and R₉ together to with the atoms to which they are attachedcan form a ring, R₇ and the ring system formed by R₈ and R₉ form anoptionally substituted 8 to 14 membered bicyclic fused ring system,wherein the bicyclic fused ring system is optionally further fused withan optionally substituted phenyl to form an optionally substituted 10 to16 membered tricyclic fused ring system.

In several embodiments, R₁ is:

wherein T is —C(O)—, and R is

In several embodiments, R₁ is a group selected from:

In some embodiments, R₁ is

where R is defined above.

Additional examples of R₁ are illustrated in PCT publications WO2004/103996 A1, WO 2004/72243 A2, WO 03/064456 A1, WO 03/64455 A2, WO03/064416 A1, and U.S. Patent Publication US 2005/0090450, as well asthose other publications referenced herein, each of which isincorporated in its entirety by reference.

2. Substituent R₂:

Each R₂ is —Z^(B)R₅, wherein each Z^(B) is independently a bond or anoptionally substituted branched or straight (C₁₋₁₂)-aliphatic chainwherein up to three carbon units of Z^(B) are optionally andindependently replaced by —C(O)—, —CS—, —C(O)NR^(B)—,—C(O)NR^(B)NR^(B)—, —C(O)O—, —NR^(B)C(O)O—, —O—, —NR^(B)C(O)NR^(B)—,—NR^(B)NR^(B)—, —NR^(B)C(O)—, —S—, —SO—, —SO₂—, —NR^(B)—, —SO₂NR^(B)—,or —NR^(B)SO₂NR^(B)—. Each R₅ is independently R^(B), halo, —OH, —CN,—NO₂, —NH₂, or —OCF₃. Each R^(B) is independently hydrogen, anoptionally substituted aliphatic, an optionally substitutedcycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl.

In several embodiments, R₂ is —Z^(B)R₅, wherein each Z^(B) isindependently a bond or an optionally substituted branched or straightC₁₋₁₂ aliphatic chain wherein up to three carbon units of Z^(B) areoptionally and independently replaced by —C(O)—, —C(S)—, —C(O)NR^(B)—,—C(O)NR^(B)NR^(B)—, —C(O)O—, —NR^(B)C(O)O—, —NR^(B)C(O)NR^(B)—,—NR^(B)NR^(B)—, —S—, —SO—, —SO₂—, —NR^(B)—, —SO₂NR^(B)—, or—NR^(B)SO₂NR^(B)—, provided that SO, SO₂, or —SO₂NR^(B)— is not directlybound to the carbonyl of formula I. Each R₅ is independently R^(B),halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃. Each R^(B) is independentlyhydrogen, an optionally substituted aliphatic, an optionally substitutedcycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl.

In still further embodiments, R₂ is —Z₁—V₁—Z₂—V₂—Z₃—V₃ each of V₁, V₂,and V₃ is independently a bond, an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, an optionallysubstituted heteroaryl, or a hydrogen when V₁, V₂, V₃ is the terminalgroup of R₂; each of Z₁, Z₂, and Z₃ is independently a bond, —C(O)—,—C(O)C(O)—, —C(S)—, —C(O)N(Q₆)-, —N(Q₆)C(O)—, —C(O)C(O)N(Q₆)-, —O—, SO—,—SO₂—, —N(Q₆)SO₂—, —N(Q₆)C(O)N(Q₆)-, —N(Q₆)C(S)N(Q₆)-, —N(Q₆)-,—N(Q₆)SO₂—, —SO₂N(Q₆)-, —C(O)N(Q₆)SO₂—, —SO₂N(Q₆)C(O)—, or hydrogen whenZ₁, Z₂, or Z₃ is the terminal group of R₂; and each Q₆ is independentlyhydrogen, or an optionally substituted aliphatic.

In other embodiments, R₂ is an optionally substituted (aliphatic)aminowherein the aliphatic portion of R₂ is —Z₂—V₂—Z₃—V₃ or —Z₃—V₃ whereineach of Z₂ and Z₃ is independently a bond, —C(O)—, —N(Q₅)-, —CH(OH)—,—C(O)N(Q₆)-, or —C(O)C(O)N(Q₆)-; V₂ is independently a bond, anoptionally substituted aliphatic, or an optionally substitutedcycloaliphatic; and V₃ is hydrogen, an optionally substituted aliphatic,or an optionally substituted cycloaliphatic.

In still further embodiments, Z₂ is —CH(OH)—, V₂ is a bond, and Z₃ is—C(O)N(Q₆)- such that R₂ is —N(Q₆)-CH(OH)—C(O)—N(V₃)(Q₆).

In certain embodiments, R₂ is an optionally substituted(aliphatic)amino, optionally substituted (cycloaliphatic)amino, anoptionally substituted alkoxy, or hydroxy.

In still another embodiment, R₂ is an alkoxy optionally substituted with1-3 of halo, hydroxy, aliphatic, cycloaliphatic, orheterocycloaliphatic.

In several embodiments, R₂ is amino. Examples of R₂ include amono-substituted amino. Additional examples of R₂ include(cycloaliphatic(carbonyl(carbonyl(alkyl))))amino(amino(carbonyl(carbonyl(aliphatic))))amino,(aliphatic(carbonyl(carbonyl(aliphatic))))amino, or(aryl(amino(carbonyl(carbonyl(aliphatic)))))amino, each of which isoptionally substituted with 1 to 3 substituents.

In several embodiments, R₂ is —NR_(2Z)R′_(2Z), —SR_(2Y), or—NR_(2Y)—CR_(2X)R′_(2X)-L₁-NR_(2Z)—R_(2W), wherein R_(2Y) isindependently hydrogen, an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl; each R_(2W) is independently hydrogen,optionally substituted aliphatic, optionally substituted aryl,optionally substituted heteroaryl, optionally substitutedheterocycloaliphatic, or optionally substituted cycloaliphatic; eachR_(2X) and R′_(2X) is independently hydrogen, an optionally substitutedaliphatic, an optionally substituted heteroaryl, an optionallysubstituted phenyl, an optionally substituted cycloaliphatic, anoptionally substituted heterocycloaliphatic; or R_(2X) and R′_(2X)together with the atom to which they are both attached form anoptionally substituted 3 to 7 membered cycloaliphatic orheterocycloaliphatic ring; each L₁ is —CH₂—, —C(O)—, —CF₂—, —C(O)C(O)—,—C(O)O—, —S(O)—, or —SO₂—; each R_(2Z) or R′_(2Z) is hydrogen, anoptionally substituted aliphatic, an optionally substitutedcycloaliphatic, an optionally substituted heterocycloaliphatic, anoptionally substituted aryl, or an optionally substituted heteroaryl; orR_(2Z) and R′_(2Z) together with the nitrogen to which they are bothattached may form an optionally substituted 3 to 7 memberedheterocycloaliphatic ring.

In several embodiments, each R_(2X) and R′_(2X) is independentlyhydrogen, or optionally substituted aliphatic, optionally substitutedcycloaliphatic, or optionally substituted (cycloaliphatic)aliphatic.

In several embodiments, L₁ is —C(O)C(O)— or —SO₂—.

In several other embodiments, each R_(2W) is hydrogen or optionallysubstituted cycloaliphatic.

In several embodiments, R₂ is —NH—CHR_(2X)—C(O)—C(O)—N(R_(2Z))R_(2W).

In several embodiments, R₂ is —NH—CHR_(2X)—CH(OH)—C(O)—N(R_(2Z))R_(2W).

In several embodiments, R₂ is —NH—CHR_(2X)—C(O)—C(O)—NHR_(2Z) wherein—NHR_(2Z) is NH-cyclopropyl, —NH-Me, —NH-Et, —NH-iPr, —NH-nPr.

In several embodiments R₂ is —NR_(2Z)R′_(2Z), —SR_(2Z) wherein eachR_(2Z) and R′_(2Z) is independently hydrogen, alkyl, cycloalkyl oraralkyl. Non-limiting examples of R_(2Z) include methyl, ethyl, t-butyl,cyclopentyl, cyclohexyl and benzyl.

In other embodiments R₂ is (—NH—CR_(2X)R′_(2X)-L₁-C(O)_(i)-M; whereineach M is independently —OH, R_(2X), —NR_(2Z)R′_(2Z), or —OR_(2X), eachi is 1 or 2, and L₁, R_(2Z), R_(2X), and R′_(2Z) are defined above.

In several embodiments R₂ is (—NH—CR_(2Z)R′_(2Z)-L₁-C(O)_(i)-M whereinL₁ is —C(O)—, i is 1 and M is independently R_(2X), —N(R_(2X)R′_(2X)),—OR_(2X), —NHSO₂R_(2X), or —SR_(2X).

In some embodiments, R′_(2Z) is H and R_(2Z) is aliphatic,(aryl)aliphatic or cycloaliphatic. Non-limiting examples of R_(2X)include hydrogen,

In some embodiments R′_(2X) is H and R_(2X) is optionally substitutedaliphatic, optionally substituted aryl, optionally substituted aralkyl,optionally substituted heteroaliphatic or optionally substitutedheteroaralkyl. Some non-limiting examples of R_(2X) include:

where c is 0-3.

In several embodiments, R₂ is:

wherein R_(2X) is

and R_(2W) is

or hydrogen.

In some embodiments, R₂ is

wherein each R₅₆ is independently optionally substituted C₁₋₆ aliphatic;optionally substituted aryl, optionally substituted heteraryl,optionally substituted cycloaliphatic, or optionally substitutedheterocycloaliphatic; each R₅₇ is independently optionally substitutedaliphatic, optionally substituted aryl, optionally substitutedaliphatic, optionally substituted heteroaryl, optionally substitutedaliphatic, optionally substituted cycloaliphatic or optionallysubstituted amino; and m is 1 or 2; and each R_(2X) and R′_(2X) isindependently hydrogen, an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl; or R_(2X) and R′_(2X) together with the atom towhich they are both attached form an optionally substituted 3 to 7membered cycloaliphatic or heterocycloaliphatic ring.

In some other embodiments, R₂ is

wherein R₅₈ and R₅₉ are each independently selected from optionallysubstituted aliphatic, optionally substituted alkoxy, optionallysubstituted aryloxy, optionally substituted heteroaryloxy, optionallysubstituted (cycloaliphatic)oxy, optionally substituted(heterocycloaliphatic)oxy optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted cycloaliphatic oroptionally substituted amino; and each R_(2X) and R′_(2X) isindependently hydrogen, an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl; or R_(2X) and R′_(2X) together with the atom towhich they are both attached form an optionally substituted 3 to 7membered cycloaliphatic or heterocycloaliphatic ring.

In several embodiments, a portion of R₁ can form cyclic structures witha portion of R₂. One non-limiting example includes:

In several embodiments, R₂ is one selected from:

In some specific embodiments, R₂ is

where X₂₀₀ is —OX₂₀₂ OR —X₂₀₂, and X₂₀₂ is aliphatic, cycloaliphatic,heterocycloaliphatic, aryl, or heteroaryl.

In other embodiments, R₂ is

Additional examples of R₂ are illustrated in PCT publications WO2004/103996 A1, WO 2004/72243 A2, WO 03/064456 A1, WO 03/64455 A2, WO03/064416 A1, and U.S. Patent Publication US 2005/0090450, as well asthose other publications referenced herein, each of which isincorporated in its entirety by reference.

3. Substituent R₃:

Each R₃ is an aliphatic, a cycloaliphatic, a heterocycloaliphatic, anaryl, or a heteroaryl, each of which is optionally substituted.

In several embodiments, each R₃ is independently —Z^(C)R₆, wherein eachZ^(C) is independently a bond or an optionally substituted branched orstraight C₁₋₆ aliphatic chain wherein up to two carbon units of Z^(C)are optionally and independently replaced by —C(O)—, —CS—, —C(O)NR^(C)—,—C(O)NR^(C)NR^(C)—, —C(O)O—, —NR^(C)C(O)O—, —O—, —NR^(C)C(O)NR^(C)—,—NR^(C)NR^(C)—, —S—, —SO—, —SO₂—, —NR^(C)—, —SO₂NR^(C)—, or—NR^(C)SO₂NR^(C)—. Each R₆ is independently R^(C), halo, —OH, —CN, —NO₂,—NH₂, or —OCF₃. Each R^(C) is independently hydrogen, an optionallysubstituted aliphatic group, an optionally substituted cycloaliphatic,an optionally substituted heterocycloaliphatic, an optionallysubstituted aryl, or an optionally substituted heteroaryl. However, inmany embodiments, when Z^(C) is a bond and R₆ is R^(C), then R^(C) isindependently an optionally substituted aliphatic group, an optionallysubstituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl.

In still other embodiments, each R₃ is an optionally substitutedaliphatic, amino, sulfonyl, sulfanyl, sulfinyl, sulfonamide, sulfamide,sulfo, —O—R_(3A), an optionally substituted cycloaliphatic, anoptionally substituted heterocycloaliphatic, an optionally substitutedaryl, or an optionally substituted heteroaryl; and each R_(3A) isindependently an optionally substituted aliphatic, an optionallysubstituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl.

In several embodiments, R₃ is an optionally substituted aryl. In someexamples, R₃ is a monocyclic, bicyclic, or tricyclic aryl, each of whichis optionally substituted. For example, R₃ is an optionally substitutedphenyl, an optionally substituted naphthyl, or an optionally substitutedanthracenyl. In other examples, R₃ is a monocyclic, bicyclic, ortricyclic aryl, each of which is optionally substituted with 1-4 ofhalo, hydroxy, cyano, nitro, aliphatic, haloaliphatic, (aliphatic)oxy,(halo(aliphatic))oxy, (aliphatic(oxy(aryl)))oxy, aryl, heteroaryl,haloaryl, cycloaliphatic, heterocycloaliphatic, or combinations thereof.In several examples, R₃ is an optionally substituted fused bicyclicaryl. In several examples, R₃ is an optionally substituted fusedtricyclic aryl.

In several embodiments, R₃ is an optionally substituted heteroaryl. Inseveral examples, R₃ is a monocyclic or bicyclic heteroaryl, each ofwhich is optionally substituted with 1-4 of halo, hydroxy, cyano, nitro,aliphatic, haloaliphatic, (aliphatic)oxy, (halo(aliphatic))oxy,(aliphatic(oxy(aryl)))oxy, aryl, heteroaryl, haloaryl, cycloaliphatic,heterocycloaliphatic, or combinations thereof.

In some embodiments R₃ is optionally substituted aliphatic such asmethyl, ethyl or propyl, each of which is optionally substituted.

According to other embodiments, R₃ is an optionally substitutedaliphatic.

According to other embodiments, R₃ is an optionally substituted(C₁₋₅)-aliphatic.

In several examples, R₃ is

In several embodiments, R₃ is one selected from:

4. Group A:

Each A is —(CX₁X₂)_(a)—, wherein each X₁ and X₂ is independentlyhydrogen, optionally substituted (C₁₋₄)-aliphatic, or optionallysubstituted aryl; or X₁ and X₂ taken together form an oxo group; andeach a is 0 to 3.

In several embodiments, X₁ or X₂ is hydrogen.

In several embodiments, X₁ or X₂ is optionally substituted(C₁₋₄)-aliphatic. Examples of X₁ or X₂ include trifluoromethyl, oroptionally substituted ethyl, propyl, butyl, or isomers thereof.

In several embodiments, X₁ or X₂ is an optionally substituted aryl.Examples of X₁ or X₂ include optionally substituted phenyl, naphthyl, orazulenyl.

5. Group B:

Each B is —(CX₁X₂)_(b)—, wherein each X₁ and X₂ is independentlyhydrogen, optionally substituted (C₁₋₄)-aliphatic, or optionallysubstituted aryl; or X₁ and X₂ taken together form an oxo group; andeach b is 0 to 3.

In several embodiments, X₁ or X₂ is hydrogen.

In several embodiments, X₁ or X₂ is optionally substituted(C₁₋₄)-aliphatic. Examples of X₁ or X₂ include trifluoromethyl, oroptionally substituted ethyl, propyl, butyl, or isomers thereof. Inseveral additional examples, X₁ or X₂ is an optionally substituted mono-or di- substituted (amino)-(C₁₋₄)-aliphatic.

In several embodiments, X₁ or X₂ is an optionally substituted aryl.Examples of X₁ or X₂ include optionally substituted phenyl, naphthyl,indenyl, or azulenyl.

6. Substituents Y and Y′

In several embodiments, each Y and Y′ is independently hydrogen,optionally substituted aliphatic, or optionally substituted aryl.

Each Y and Y′ is independently —Z^(D)R₇, wherein each Z^(D) isindependently a bond or an optionally substituted straight or branched(C₁₋₆)-aliphatic chain wherein up to two carbon units of Z^(D) areoptionally and independently replaced by —C(O)—, —CS—, —C(O)NR^(D)—,—C(O)NR^(D)NR^(D)—, —C(O)O—, —OC(O)—, —NR^(D)C(O)O—, —O—,—NR^(D)C(O)NR^(D)—, —OC(O)NR^(D)—, —NR^(D)NR^(D)—, —NR^(D)C(O)—, —S—,—SO—, —SO₂—, —NR^(D)—, —SO₂NR^(D)—, —NR^(D)SO₂—, or —NR^(D)SO₂NR^(D)—.Each R₇ is independently R^(D), halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃.Each R^(D) is independently hydrogen, or optionally substituted aryl.

In several embodiments, one selected from Y and Y′ is hydrogen.

In several embodiments, one selected from Y and Y′ is optionallysubstituted aliphatic.

In several embodiments, one selected from Y and Y′ is optionallysubstituted aryl.

In several embodiments, both Y and Y′ are hydrogen.

In several embodiments, one of Y or Y′ is hydrogen and the other isfluorine.

In several embodiments, both of Y and Y′ are fluorine.

In additional of examples, one of Y or Y′ is hydrogen and the other ismethoxycarbonyl; one of Y or Y′ is hydrogen and the other is hydroxy; ortogether, Y and Y′ form an oxo group or form ═S.

7. Exceptions:

In compounds of formula (I), a+b is 2 or 3. For example, a is 0 and b is3; a is 1 and b is 2; a is 2 and b is 1; or a is 3 and b is 0.

C. Sub-generic Compounds:

Another aspect of the present invention provides compounds of formula Iauseful for inhibiting serine protease activity and methods inhibitingserine protease activity. Compounds of formula Ia include:

or a pharmaceutically acceptable salt thereof wherein R₃, A, B, Y, andY′ are defined above in formula I.

Each R_(1a) is -Q₄-W₄-Q₃-W₃-Q₂-W₂-Q₁; wherein each of W₂, W₃, and W₄ isindependently a bond, —C(O)—, —C(S)—, —C(O)N(Q₅)-, —C(O)O—, —O—,—N(Q₅)C(O)N(Q₅)-, —SO₂—, —N(Q₅)SO₂—, —S—, —N(Q₅)-, —SO—, —N(Q₅)C(O)—,—OC(O)—, —N(Q₅)C(O)O—, or —SO₂N(Q₅)-; each of Q₁, Q₂, Q₃ and Q₄ isindependently a bond, an optionally substituted C₁₋₄ aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, an optionallysubstituted heteroaryl, or a hydrogen when Q₁, Q₂, Q₃, or Q₄ is theterminal group of R₁; and each Q₅ is independently hydrogen or anoptionally substituted aliphatic.

Each R_(2a) is —Z₁—V₁—Z₂—V₂—Z₃—V₃ each of V₁, V₂, and V₃ isindependently a bond, an optionally substituted aliphatic, an optionallysubstituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, an optionallysubstituted heteroaryl, or a hydrogen when V₁, V₂, V₃ is the terminalgroup of R₂; each of Z₁, Z₂, and Z₃ is independently a bond, —C(O)—,—C(O)C(O)—, —C(S)—, —C(O)N(Q₅)-, —N(Q₅)C(O)—, —C(O)C(O)N(Q₅)-, —O—, SO—,—SO₂—, —N(Q₅)SO₂—, —N(Q₅)C(O)N(Q₅)-, —N(Q₅)C(S)N(Q₅)-, —N(Q₅)-,—N(Q₅)SO₂—, —SO₂N(Q₅)-, —C(O)N(Q₅)SO₂—, —SO₂N(Q₅)C(O)—, or hydrogen whenZ₁, Z₂, or Z₃ is the terminal group of R₂; and each Q₅ is independentlyhydrogen, or an optionally substituted aliphatic.

In several examples, R_(2a) is an optionally substituted(aliphatic)amino, an optionally substituted alkoxy, or hydroxy.

In several examples, R_(2a) is an (aliphatic)amino wherein the nitrogenatom is optionally substituted with —Z₂—V₂—Z₃—V₃ or —Z₃—V₃ wherein eachof Z₂ and Z₃ is independently a bond, —C(O)—, —N(Q₅)-, or—C(O)C(O)N(Q₅)-; and each of V₂ and V₃ is independently a bond, anoptionally substituted aliphatic, or an optionally substitutedcycloaliphatic.

Another aspect of the present invention provides compounds of formula Ibuseful for inhibiting serine protease activity and methods inhibitingserine protease activity. Compounds of formula Ib include:

or a pharmaceutically acceptable salt thereof, wherein R₃, R₈, R, T, A,B, Y and Y′ are defined above in formula I.

Each G is a 2 to 15 atom optionally substituted aliphatic chainoptionally containing 1 to 3 heteroatoms selected from O, S and N.

Examples of compounds of formula Ib include:

wherein T, R, and R₃ are defined above in formula I.

Still other examples of formula Ib are

wherein each R_(2W) is independently

or hydrogen; each T is independently a bond, —C(O)—, —OC(O)—, —NHC(O)—,—S(O)₂N(H)—, —C(O)C(O)— or —SO₂—; each R is independently hydrogen, anoptionally substituted aliphatic, an optionally substitutedcycloaliphatic, an optionally substituted heterocycloaliphatic, anoptionally substituted aryl, or an optionally substituted heteroaryl;and each R₉ is independently hydrogen, an optionally substitutedaliphatic, an optionally substituted heteroaryl, an optionallysubstituted phenyl.

Further specific examples of compounds of formula Ib are

Other examples of compounds of formula Ib include:

Another aspect of the present invention provides compounds of formula IIuseful for inhibiting serine protease activity and methods inhibitingserine protease activity. Compounds of formula II include:

or a pharmaceutically acceptable salt thereof, wherein

Each R₃ is an optionally substituted aryl or an optionally substitutedheteroaryl;

Each R_(2Y) is independently hydrogen, an optionally substitutedaliphatic, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl;

Each R₉ is independently hydrogen, optionally substituted aliphatic,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocycloaliphatic, or optionally substitutedcycloaliphatic;

Each R_(2X) and R′_(2X) is independently hydrogen, an optionallysubstituted aliphatic, an optionally substituted heteroaryl, anoptionally substituted phenyl, an optionally substituted cycloaliphatic,an optionally substituted heterocycloaliphatic; or R_(2X) and R′_(2X)together with the atom to which they are both attached form anoptionally substituted 3 to 7 membered cycloaliphatic orheterocycloaliphatic ring, or R_(2X) and R_(2Y) together with the atomsto which they are attached form an optionally substituted 5 to 7membered heterocycloaliphatic ring;

Each R_(1b) is —Z^(E)R₂₁, wherein Z^(E) is —CH₂—, —NH—, —CH(R_(1Z))—, or—O—, and R₂₁ is optionally substituted 6-7 membered cycloaliphatic oroptionally substituted tert-butyl;

Each R_(1Z) is optionally substituted aliphatic, optionally substitutedcycloaliphatic, optionally substituted heterocycloaliphatic, optionallysubstituted aryl , or optionally substituted heteroaryl;

Each R_(2Z) is hydrogen, optionally substituted cycloaliphatic,optionally substituted heterocycloaliphatic, or optionally substitutedaliphatic; and

Each R_(2W) is hydrogen, optionally substituted cycloaliphatic,optionally substituted heterocycloaliphatic, or optionally substitutedaliphatic, or R_(2Z) and R_(2W), together with the nitrogen atom towhich they are attached form an optionally substitutedheterocycloaliphatic.

Another aspect of the present invention provides compounds of formulaIII useful for inhibiting serine protease activity and methodsinhibiting serine protease activity. Compounds of formula III include:

or a pharmaceutically acceptable salt thereof, wherein R_(1e) is

R_(2e) is

R′_(2e) is

or hydrogen; and

R_(3e) is optionally substituted aryl or optionally substitutedheteroaryl.

Another aspect of the present invention provides compounds of formula IVuseful for inhibiting serine protease activity and methods inhibitingserine protease activity. Compounds of formula IV include:

or a pharmaceutically acceptable salt thereof, wherein R_(1e) is

R_(2e) is

R′_(2e)

is or hydrogen; and

Each of R_(3f) and R′_(3f) is independently hydrogen, sulfonamide,sulfonyl, sulfinyl, optionally substituted acyl, optionally substitutedaliphatic, optionally substituted cycloaliphatic, optionally substitutedheterocycloaliphatic, optionally substituted aryl, or optionallysubstituted heteroaryl, or R_(3f) and R′_(3f) together with the nitrogenatom to which they are attached form an optionally substituted,saturated, partially unsaturated, or full unsaturated, 5-8 memberedheterocycloaliphatic or heteroaryl.

Another aspect of the present invention provides compounds of formula Vuseful for inhibiting serine protease activity and methods inhibitingserine protease activity. Compounds of formula V include:

or a pharmaceutically acceptable salt thereof, wherein R_(1e), R_(2e),and R′_(2e) are defined above in formula III.

Each D is independently —CR₈—, N, S, or O, provided that no more thantwo D are independently, S, or O, and R₈ is defined above in formula I.

Another aspect of the present invention provides compounds of formula VIuseful for inhibiting serine protease activity and methods inhibitingserine protease activity. Compounds of formula VI include:

or a pharmaceutically acceptable salt thereof, wherein R_(1e), R_(2e),and R′_(2e) are defined above in formula III.

Each R_(3g) is a substituted aryl or a substituted heteroaryl. In someembodiments, R_(3g) is

Another aspect of the present invention provides compounds of formulaVII useful for inhibiting serine protease activity and methodsinhibiting serine protease activity. Compounds of formula VII include:

or a pharmaceutically acceptable salt thereof, wherein R_(1e), R_(2e),and R′_(2e) are defined above in formula III, and R_(3g) is defined informula VI.

Another aspect of the present invention provides compounds of formulaVIII useful for inhibiting serine protease activity and methodsinhibiting serine protease activity. Compounds of formula VIII include:

or a pharmaceutically acceptable salt thereof, wherein R_(1e), R_(2e),and R′_(2e) are defined above in formula III, and R_(3g) is defined informula VI.

Another aspect of the present invention provides compounds of formula IXuseful for inhibiting serine protease activity and methods inhibitingserine protease activity. Compounds of formula IX include:

or a pharmaceutically acceptable salt thereof, wherein R_(1e), R_(2e),and R′_(2e) are defined above in formula III, and R_(3g) is defined informula VI.

Another aspect of the present invention provides compounds of formula Xuseful for inhibiting serine protease activity and methods inhibitingserine protease activity. Compounds of formula X include:

or a pharmaceutically acceptable salt thereof, wherein R_(1e), R_(2e),and R′_(2e) are defined above in formula III, and R_(3g) is defined informula VI.

D. Combinations of Embodiments

Other embodiments include any combination of the aforementionedsubstituents R₁, R₂, R₃, A, B, Y, and Y′.

E. Exemplary Compounds

The invention is intended to include compounds wherein R₁ and R₂ containstructural elements of a serine protease inhibitor. Compounds having thestructural elements of a serine protease inhibitor include, but are notlimited to, the compounds of the following publications: WO 97/43310, US20020016294, WO 01/81325, WO 01/58929, WO 01/32691, WO 02/08198, WO01/77113, WO 02/08187, WO 02/08256, WO 02/08244, WO 03/006490, WO01/74768, WO 99/50230, WO 98/17679, WO 02/48157, WO 02/08251, WO02/07761, WO 02/48172, WO 02/08256, US 20020177725, WO 02/060926, US20030008828, WO 02/48116, WO 01/64678, WO 01/07407, WO 98/46630, WO00/59929, WO 99/07733, WO 00/09588, US 20020016442, WO 00/09543, WO99/07734, U.S. Pat. Nos. 6,018,020, 6,265,380, 6,608,027, US20020032175, US 20050080017, WO 98/22496, WO 05/028502, U.S. Pat. No.5,866,684, WO 02/079234, WO 00/31129, WO 99/38888, WO 99/64442, WO2004072243, WO 02/18369, US2006046956, US2005197301, WO2005058821,WO2005051980, WO2005030796, WO2005021584, WO2005113581, WO2005087731,WO2005087725, WO2005087721, WO2005085275, WO2005085242, US2003216325,WO2003062265, WO2003062228, WO2002008256, WO 2002008198, WO2002008187,WO 2002048172, WO 2001081325, WO 2001077113, U.S. Pat. Nos. 6,251,583,5,990,276, US20040224900, US20040229818, WO2004037855, WO2004039833,WO200489974, WO2004103996, WO2004030670, WO2005028501, WO2006007700,WO2005070955, WO2006007708, WO2006000085, WO2005073195, WO2005073216,WO2004026896, WO2004072243, WO2004113365, WO2005010029, US20050153877,WO2004093798, WO2004094452, WO2005046712, WO2005051410, WO2005054430,WO2004032827, WO2005095403, WO2005077969, WO2005037860, WO2004092161,WO2005028502, WO2003087092, and WO2005037214, each of which isincorporated herein by reference.

Specific exemplary compounds of the invention are shown below in TableA.

TABLE A Exemplary compounds of Forrnula I.

 1

 2

 3

 4

 5

 6

 7

 8

 9

 10

 11

 12

 13

 14

 15

 16

 17

 18

 19

 20

 21

 22

 23

 24

 25

 26

 27

 28

 29

 30

 31

 32

 33

 34

 35

 36

 37

 38

 39

 40

 41

 42

 43

 44

 45

 46

 47

 48

 49

 50

 51

 52

 53

 54

 55

 56

 57

 58

 59

 60

 61

 62

 63

 64

 65

 66

 67

 68

 69

 70

 71

 72

 73

 74

 75

 76

 77

 78

 79

 80

 81

 82

 83

 84

 85

 86

 87

 88

 89

 90

 91

 92

 93

 94

 95

 96

 97

 98

 99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

464

465

466

467

468

469

470

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

493

494

495

496

497

498

499

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

585

586

587

588

589

590

591

592

593

594

DETAILED DESCRIPTION OF THE INVENTION

II. Synthesis of the Compounds

Compounds of Formula I may be readily synthesized from commerciallyavailable starting materials using the exemplary synthetic routesprovided below. Exemplary synthetic routes to produce compounds ofFormula I are provided below in the Preparations, Methods, Examples, andSchemes. For example, the spiroisoxazoline moiety may be prepared by1,3-dipolar addition between a nitrile oxide and a methylene proline asreported by Kurth, M. J., et. al., in J. Org. Chem., 2002, 67, pp.5673-5677, and as illustrated in Scheme 1 below. The nitrile oxides canbe generated from cholooximes or nitroalkanes using known methods.

Scheme I provides a general representation of processes for preparingcompounds of Formula I. Its overall strategy is to construct a compoundof formula 1h followed by selective removal of the protecting group PG₁in the presence of PG₂ to provide the intermediate 1j. The substituentR₁ may then be coupled to 1j, which provides intermediates of formula 1kcontaining R₁. In some embodiments, R₁ may itself contain a protectinggroup which may be selectively removed in the presence of PG₂, followedby further elaboration. Subsequent to the addition of the R₁ moiety, thePG₂ group is removed to provide the intermediate 1m. Coupling of 1m withan R₂ moiety then provides the peptidomimetic compounds of Formula I.

Referring again to Scheme 1, in one example, the hydroxy proline 1a isprotected as the Boc derivative (i.e., step ia) to provide the protectedproline 1b, wherein PG₁ is t-butyloxycarbonate, using known methods.See, e.g., T. W. Greene and P. G. M. Wuts, Protective Groups in OrganicSynthesis, 3^(rd) edition, John Wiley and Sons, Inc. (1999). Oxidationof 1b (i.e., step ib) provides the keto-pyrrolidine acid 1c. Theoxidation is achieved with a suitable reagent such as, for example,sodium hypochlorite in the presence of TEMPO. Next, in step ic, theketo-pyrrolidine acid 1c is reacted with a Wittig reagent such as, forexample, a triphenylphosphonium ylid of the formula (Ph)₃P═C(Y)(Y′) andusing known conditions, to provide an exomethylene compound of formula1d. Use of the free acid 1c to provide the corresponding free acid 1d isadvantageous as the acid 1d may be expediently purified from neutral orbasic by-products by simple extraction of 1d into aqueous basicsolution. The acid 1d is subsequently protected (step id) with asuitable protecting group such as, for example, a t-butyl ester underknown conditions (ibid) to provide the intermediate 1e.

Reaction of 1e with a nitrile oxide 1f provides a mixture of the syn andanti isomers of the spiroisoxazolines 1g and 1h. As referred to herein,syn- means that the 2-carboxyl moiety of the proline ring and the oxygenof the isoxazoline ring are on the same side of a plane as described bythe proline ring. The term anti- means that the 2-carboxyl moiety of theproline ring and the oxygen of the isoxazoline ring are on the oppositeside of a plane as described by the proline ring. Thus, 1g represents asyn-compound of the invention and 1h represents an anti-compound of theinvention.

In some embodiments, when PG₁ is Boc and PG₂ is t-butoxy, selectiveremoval of the protecting group PG₁ from 1g and 1h in the presence ofthe protecting group PG₂ may be achieved with a sulfonic acid such as,for example, methane sulfonic acid in a suitable organic solvent attemperatures from about −40° C. to about 40° C., from about −20° C. toabout 20° C. and from about −5° C. to about 5° C. Suitable organicsolvents include, for example, methylene chloride and tetrahydrofuran.

The isomers 1i and 1j may be separated advantageously by crystallizationof a mixture of the corresponding organic acid salts which avoids morecomplicated methods such as, e.g., chromatography. Suitable organicsalts include those of organic carboxylic acids, e.g., acetic acid,optionally substituted benzoic acids, tartaric acid, malonic acid,fumaric acid, oxalic acid, mandelic acid, citric acid, p-toluoyltartaric acid and maleic acid; organic sulfonic acids, e.g., methanesulfonic acid, optionally substituted benzene sulfonic acids,trifluoromethane sulfonic acid and camphor sulfonic acid.

A single spiroisoxazoline isomer, for example 1j, is coupled with anacid R₁COOH in the presence of a coupling reagent such as, for example,EDCI to provide the intermediate spiroisoxazoline 1k. Selective removalof the protecting group PG₂ of 1k to give 1m with minimum racemizationor cleavage of the R₁ side chain is achieved by a suitable mineral acidin a suitable organic solvent at temperatures from about −40° C. toabout 40° C., from about −20° C. to about 20° C. and from about −5° C.to about 5° C. Suitable mineral acids include, for example, concentratedhydrochloric acid or concentrated sulfuric acid. Suitable organicsolvents include, for example, methylene chloride and tetrahydrofuran.The spiroisoxazoline 1m is then coupled with an amine moiety R₂ toprovide the compounds of Formula I.

Referring again to Scheme 1, PG₁(CO)— can be an amine protecting group,wherein PG₁ is, for example, methoxycarbonyl, t-butyloxycarbonyl,9-flourenylmethyloxycarbonyl, or benzyloxycarbonyl. PG₂(CO)— can be anacid or acid protecting group wherein PG₂ is, for example, —OH, methoxy,t-butyloxy or benzyloxy.

Each of PG₁ and PG₂ groups may be incorporated into the corespiroisoxazoline structure either individually or together using knownmethods and as further described herein. For example, if the desired R₁substituted is a group other than a PG₁ group (e.g., a protectinggroup), the PG₁ group may be removed to provide a compound with a freeamine group. That amine group and an appropriate moiety may be coupledunder known coupling conditions to provide a compound wherein R₁ is amoiety of a protease inhibitor. For example, if the PG₂ moiety isprotected, the protecting group may be removed and an R₂ moiety may beincorporated.

Another method for producing compounds of the present invention isillustrated below in Scheme 2.

Referring to Scheme 2, the symbol

represents a polymeric resin to which reactants are bound by afunctionality that allows further modification and subsequent removal ofthe product from the resin. A suitable resin is a polymer bounddihydropyran (DHP) resin as described by Ellman et. al. in TetrahedronLetters, 1994, 35, 9333.

In step iia, simultaneous deprotection of both the amine and acid may beachieved by contacting the proline 1e with an acid, for example,trifluoroacetic acid in methylene chloride to give the amino acid 2a.Reaction of 2a, step iib, with an activated Fmoc derivative, forexample, N-(9H-Fluoren-9ylmethoxycarbonyloxy)succinimide (Fmoc-OSu), inthe presence of a mild inorganic base, such as sodium carbonate, givesthe Fmoc derivative 2b.

Preparation of the resin bound peptide 2d may be accomplished byreacting the Fmoc derivative 2b with the DHP resin bound amino-alcohol2c, step iiic, which reacts with the free acid 2b, in the presence of acoupling reagent such as, for example,O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU), a racemization suppressant, such as 1-hydroxybenzotriazole(HOBT) and a tertiary amine, such as di-isopropylethyl amine (DIEA).

As in Scheme 1, an R₃-substituted nitrile oxide 1f may undergo a dipolarcycloaddition reaction with the resin bound peptide 2d to provide twoisomers, syn- and anti-, of the compound 2e. Next in step iid, the Fmocprotecting group is removed by contacting 2e with a secondary amine suchas, for example, piperidine in a polar solvent such as dimethylformamideto give 2f. Formation of the peptide 2g, via step iie, can be achievedthrough reaction of 2f with a carboxylic acid in the presence of acoupling reagent such as HBTU, a racemization suppressant such as HOBt,and a tertiary amine such as DIEA. Cleavage of the peptide-resin 2g,step iif, to give the alpha-hydroxy-amide 2h, can be achieved bycontacting 2g with a strong acid such as, for example, trifluoroaceticacid and water.

In the final step, iig, the alpha-hydroxy-amide 2h is oxidized to 2iusing a Dess-Martin periodinane oxidation or a Pfitzner-Moffatoxidation.

Alternatively, compounds of Formula I may be prepared using resin boundreagents as illustrated below in Scheme 3.

In Scheme 3, the selective removal of the PG₁ in the presence of PG₂(step if) provides spiroisoxazoline isomer(s) 1i and/or 1j. Reaction of1i and/or 1j, in step iiia, with an activated Fmoc derivative, e.g.,N-(9H-Fluoren-9-ylmethoxycarbonyloxy)succinimide (Fmoc-OSu), in thepresence of a mild inorganic base, such as sodium carbonate, providesthe Fmoc derivative 3a.

Preparation of the resin bound peptide 2e may be accomplished byreaction of the Fmoc derivative 3a with the DHP resin boundamino-alcohol 2c, via step iiib, which reacts with a free acid 3b, inthe presence of a coupling reagent (e.g.,O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU)), a racemization suppressant (e.g., 1-hydroxybenzotriazole(HOBT)), and a tertiary amine (e.g., di-isopropylethyl amine (DIEA)).

In step iid, the Fmoc protecting group is removed by contacting 2e witha secondary amine such as, e.g., piperidine in a polar solvent such asdimethylformamide to give 2f. Formation of the peptide 2g can beachieved, e.g., by reacting 2f with a carboxylic acid in the presence ofa coupling reagent (e.g., HBTU), a racemization suppressant (e.g., HOBt)and a tertiary amine (e.g., DIEA). Cleavage of the peptide-resin 2g togive the free peptide 2h can be achieved, e.g., by contacting 2g with astrong acid (e.g., trifluoroacetic acid) and water.

In the final step, iig, the alcohol of 2h can be oxidized to 2i, e.g.,with Dess-Martin periodinane or sodium hypochlorite and TEMPO.

Scheme 4 below illustrates a synthetic pathway for compounds of FormulaI in which R₁ and R₂, together with the atoms to which they areattached, form an optionally substituted macrocyclicheterocycloaliphatic.

Referring to Scheme 4, the spiroisoxazoline acid E4 reacts with theamino ester H1 in the presence of a coupling reagent to provide theintermediate H2. Macrocyclization of H2 results in compound H3.Hydrolysis of the ester H2 provides acid H4. Reaction of acid H4 with asulfonamide or sulfamide in the presence of a coupling reagent providesthe product H5.

Shown below in Schemes 5, 6, 7, 8, and 9 are examples of total synthesisof compounds of Formula I according to one of the methods describedabove.

Referring to Scheme 5, the protectedt-butyldimethylsilyl-hydroxybenzaldehyde 5b is converted to thehydroxamoyl chloride 5d as previously described. Reaction of 5d with theexomethylene pyrrolidine provides the spiroisoxazoline 5e. Deprotectionof 5e to 5f followed by reaction with triflic anhydride provides thetriflate 5g. Reaction of 5f with an amine HNU₁U₂ provides theintermediate spiroisoxazoline 5h which is converted to compounds of theinvention as previously described.

Alternatively, the hydroxy-spiroisoxazoline intermediate 5f may bealkylated to provide the intermediate 5k which may be similarlyconverted to compounds of the invention.

Referring to Scheme 6, reaction of the diprotected pyrrolidinone withdifluorodibromomethane in the presence of HMPT and zinc provides thedifluroexomethylene intermediate 6b. Dipolar addition with the nitrileoxide if as previously described provides the diflurospiroisoxazoline6c. In a similar fashion, the intermediates 6b and 6f are prepared from6a and 6e respectively and converted to the corresponding substitutedisooxazolines 6d and 6g.

In other variations, the intermediate 6h may be brominated to give 6j,alkylated to provide 6k or oxidized to provide 6m using the reagentsillustrated.

Referring to Scheme 7, dipolar addition of the exomethylene pyrrolidineshown with if wherein R₃ is —COOEt, leads to the ester 7a. Hydrolysis ofthe ethyl ester in 7a, conversion to the acid chloride (not shown) andreaction with ammonia provides the amide 7c. Reaction of 7c withtrifluoroacetic anhydride provides the nitrile 7d which is converted tothe peptidic intermediate 7e by methods previously described. Theintermediate 7e reacts with an azide U₄N₃ to provide the tetrazole 7fwhich is oxidized to a compound of the invention 7g. In a variation ofthis scheme, the ester 7a may be converted to the triazole 7h andsubsequently to compounds of the invention 7i.

Referring to Scheme 8, dipolar addition as previously described butusing hydroxycarbonimidic dibromide provides the bromoisoxazoline 8a.Reaction of 8a with an arylboronic acid in the presence of a palladiumcatalyst (Suzuki conditions) provides the intermediate 8b which isconverted to compounds of the invention by methods previously described.The AR in step 8a and 8b represents aryl or heteroaryl.

Referring to Scheme 9, the Wittig product 9a undergoes a dipolaraddition to provide the spiroisoxazoline 9b. Reduction of 9b with, forexample, DIBAL provides the alcohol 9c which may be alkylated to providethe intermediate 9e which subsequently may be converted to compounds ofthe invention by methods previously described. Hydrolysis of ester 9bwith, e.g., LiOH, will provide carboxylic acid 9d which can be convertedto compounds of formula I as described herein.

Referring to scheme 10, the diprotected piperidinone 10b undergoes aWittig type reaction to form the exomethylene compound 10c whichundergoes dipolar addition as previously described to provide a 4.5spiroisoxazoline 10d which may be converted to compounds of theinvention as previously described.

III. Formulations, Administrations, and Uses

Another embodiment of this invention provides a pharmaceuticalcomposition comprising a compound of Formula I or pharmaceuticallyacceptable salts or mixtures of salts thereof. According to anotherembodiment, the compound of Formula I is present in an amount effectiveto decrease the viral load in a sample or in a patient, wherein saidvirus encodes a serine protease necessary for the viral life cycle, anda pharmaceutically acceptable carrier.

If pharmaceutically acceptable salts of the compounds of this inventionare utilized in these compositions, those salts are preferably derivedfrom inorganic or organic acids and bases. Included among such acidsalts are the following: acetate, adipate, alginate, aspartate,benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate,camphor sulfonate, cyclopentane-propionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2 hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2 naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3 phenyl propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.Base salts include ammonium salts, alkali metal salts, such as sodiumand potassium salts, alkaline earth metal salts, such as calcium andmagnesium salts, salts with organic bases, such as dicyclohexylaminesalts, N methyl D glucamine, and salts with amino acids such asarginine, lysine, and so forth.

Also, the basic nitrogen containing groups may be quaternized with suchagents as lower alkyl halides, such as methyl, ethyl, propyl, and butylchloride, bromides and iodides; dialkyl sulfates, such as dimethyl,diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkylhalides, such as benzyl and phenethyl bromides and others. Water or oilsoluble or dispersible products are thereby obtained.

The compounds utilized in the compositions and methods of this inventionmay also be modified by appending appropriate functionalities to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological system (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

Pharmaceutically acceptable carriers that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes, polyethylenepolyoxypropylene block polymers, polyethylene glycol and wool fat.

According to another embodiment, the compositions of this invention areformulated for pharmaceutical administration to a mammal. In oneembodiment said mammal is a human being.

Such pharmaceutical compositions of the present invention may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir.The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra articular, intra synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. Preferably, the compositions are administeredorally or intravenously.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non toxicparenterally acceptable diluent or solvent, for example as a solution in1,3 butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or diglycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

In one embodiment, dosage levels of between about 0.01 and about 100mg/kg body weight per day of the protease inhibitor compounds describedherein are useful in a monotherapy for the prevention and treatment ofantiviral, particularly anti-HCV mediated disease. In anotherembodiment, dosage levels of between about 0.5 and about 75 mg/kg bodyweight per day of the protease inhibitor compounds described herein areuseful in a monotherapy for the prevention and treatment of antiviral,particularly anti-HCV mediated disease. Typically, the pharmaceuticalcompositions of this invention will be administered from about 1 toabout 5 times per day or alternatively, as a continuous infusion. Suchadministration can be used as a chronic or acute therapy. The amount ofactive ingredient that may be combined with the carrier materials toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration. A typical preparation willcontain from about 5% to about 95% active compound (w/w). In oneembodiment, such preparations contain from about 20% to about 80% activecompound.

When the compositions of this invention comprise a combination of acompound of formula I and one or more additional therapeutic orprophylactic agents, both the compound and the additional agent shouldbe present at dosage levels of between about 10 to 100% of the dosagenormally administered in a monotherapy regimen. In another embodiment,the additional agent should be present at dosage levels of between about10 to 80% of the dosage normally administered in a monotherapy regimen.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers that are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried cornstarch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These may be prepared by mixing the agent with a suitable non irritatingexcipient which is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract may be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions may be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith our without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

In one embodiment, the pharmaceutical compositions are formulated fororal administration.

In another embodiment, the compositions of this invention additionallycomprise another anti-viral agent, preferably an anti-HCV agent. Suchanti-viral agents include, but are not limited to, immunomodulatoryagents, such as α, β-, and γ-interferons, pegylated derivatizedinterferon-α compounds, and thymosin; other anti-viral agents, such asribavirin, amantadine, and telbivudine; other inhibitors of hepatitis Cproteases (NS2-NS3 inhibitors and NS3-NS4A inhibitors); inhibitors ofother targets in the HCV life cycle, including helicase and polymeraseinhibitors; inhibitors of internal ribosome entry; broad-spectrum viralinhibitors, such as IMPDH inhibitors (e.g., compounds of U.S. Pat. Nos.5,807,876, 6,498,178, 6,344,465, and 6,054,472, WO 97/40028, WO98/40381, WO 00/56331, and mycophenolic acid and derivatives thereof,and including, but not limited to VX-497, VX-148, and/or VX-944); orcombinations of any of the above. See also W. Markland et al.,Antimicrobial & Antiviral Chemotherapy, 44, p. 859 (2000) and U.S. Pat.No. 6,541,496.

The following definitions are used herein (with trademarks referring toproducts available as of this application's filing date).

-   “Peg-Intron” means PEG-INTRON®, peginteferon alfa-2b, available from    Schering Corporation, Kenilworth, N.J.;-   “Intron” means INTRON-A®, interferon alfa-2b available from Schering    Corporation, Kenilworth, N.J.;-   “ribavirin” means ribavirin    (1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available    from ICN Pharmaceuticals, Inc., Costa Mesa, Calif.; described in the    Merck Index, entry 8365, Twelfth Edition; also available as    REBETROL® from Schering Corporation, Kenilworth, N.J., or as    COPEGASUS® from Hoffmann-La Roche, Nutley, N.J.;-   “Pagasys” means PEGASYS®, peginterferon alfa-2a available    Hoffmann-La Roche, Nutley, N.J.;-   “Roferon” mean ROFERON®, recombinant interferon alfa-2a available    from Hoffmann-La Roche, Nutley, N.J.;-   “Berefor” means BEREFOR®, interferon alfa 2 available from    Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, Conn.;-   SUMIFERON®, a purified blend of natural alpha interferons such as    Sumiferon available from Sumitomo, Japan;-   WELLFERON®, interferon alpha n1 available from Glaxo_Wellcome LTd.,    Great Britain; and-   ALFERON®, a mixture of natural alpha interferons made by Interferon    Sciences, and available from Purdue Frederick Co., CT.

The term “interferon” as used herein means a member of a family ofhighly homologous species-specific proteins that inhibit viralreplication and cellular proliferation, and modulate immune response,such as interferon alpha, interferon beta, or interferon gamma. TheMerck Index, entry 5015, Twelfth Edition.

According to one embodiment of the present invention, the interferon isα-interferon. According to another embodiment, a therapeutic combinationof the present invention utilizes natural alpha interferon 2a. Or, thetherapeutic combination of the present invention utilizes natural alphainterferon 2b. In another embodiment, the therapeutic combination of thepresent invention utilizes recombinant alpha interferon 2a or 2b. In yetanother embodiment, the interferon is pegylated alpha interferon 2a or2b. Interferons suitable for the present invention include:

(a) INTRON-A® (interferon-alpha 2B, Schering Plough),

(b) PEG-INTRON®,

(c) PEGASYS®,

(d) ROFERON®,

(e) BEREFOR®,

(f) SUMIFERON®,

(g) WELLFERON®,

(h) consensus alpha interferon available from Amgen, Inc., Newbury Park,Calif.,

(i) ALFERON®;

(j) VIRAFERON®;

(k) INFERGEN®;

(l) ALBUFERON™.

As is recognized by skilled practitioners, a protease inhibitor would bepreferably administered orally. Interferon is not typically administeredorally. Nevertheless, nothing herein limits the methods or combinationsof this invention to any specific dosage forms or regime. Thus, eachcomponent of a combination according to this invention may beadministered separately, together, or in any combination thereof.

In one embodiment, the protease inhibitor and interferon areadministered in separate dosage forms. In one embodiment, any additionalagent is administered as part of a single dosage form with the proteaseinhibitor or as a separate dosage form. As this invention involves acombination of compounds, the specific amounts of each compound may bedependent on the specific amounts of each other compound in thecombination. As recognized by skilled practitioners, dosages ofinterferon are typically measured in IU (e.g., about 4 million IU toabout 12 million IU).

Accordingly, agents (whether acting as an immunomodulatory agent orotherwise) that may be used in combination with a compound of thisinvention include, but are not limited to, Albuferon™(albumin-Interferon alpha) available from Human Genome Sciences;PEG-INTRON® (peginterferon alfa-2b, available from Schering Corporation,Kenilworth, N.J.); INTRON-A®, (interferon alfa-2b available fromSchering Corporation, Kenilworth, N.J.); ribavirin(1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available fromICN Pharmaceuticals, Inc., Costa Mesa, Calif.; described in the MerckIndex, entry 8365, Twelfth Edition); REBETROL® (Schering Corporation,Kenilworth, N.J.), COPEGUS® (Hoffmann-La Roche, Nutley, N.J.); PEGASYS®(peginterferon alfa-2a available Hoffmann-La Roche, Nutley, N.J.);ROFERON® (recombinant interferon alfa-2a available from Hoffmann-LaRoche, Nutley, N.J.); BEREFOR® (interferon alfa 2 available fromBoehringer Ingelheim Pharmaceutical, Inc., Ridgefield, Conn.);SUMIFERON® (a purified blend of natural alpha interferons such asSumiferon available from Sumitomo, Japan); WELLFERON® (interferon alphan1 available from Glaxo Wellcome Ltd., Great Britain); ALFERON® (amixture of natural alpha interferons made by Interferon Sciences, andavailable from Purdue Frederick Co., CT); α-interferon; natural alphainterferon 2a; natural alpha interferon 2b; pegylated alpha interferon2a or 2b; consensus alpha interferon (Amgen, Inc., Newbury Park,Calif.); VIRAFERON®; INFERGEN®; REBETRON® (Schering Plough,Interferon-alpha 2B+Ribavirin); pegylated interferon alpha (Reddy, K. R.et al. “Efficacy and Safety of Pegylated (40-kd) Interferon alpha-2aCompared with Interferon alpha-2a in Noncirrhotic Patients with ChronicHepatitis C (Hepatology, 33, pp. 433-438 (2001); consensus interferon(Kao, J. H., et al., “Efficacy of Consensus Interferon in the Treatmentof Chronic Hepatitis” J. Gastroenterol. Hepatol. 15, pp. 1418-1423(2000); lymphoblastoid or “natural” interferon; interferon tau(Clayette, P. et al., “IFN-tau, A New Interferon Type I withAntiretroviral activity” Pathol. Biol. (Paris) 47, pp. 553-559 (1999);interleukin-2 (Davis, G. L. et al., “Future Options for the Managementof Hepatitis C.” Seminars in Liver Disease, 19, pp. 103-112 (1999);Interleukin-6 (Davis et al. “Future Options for the Management ofHepatitis C.” Seminars in Liver Disease, 19, pp. 103-112 (1999);interleukin-12 (Davis, G. L. et al., “Future Options for the Managementof Hepatitis C.” Seminars in Liver Disease, 19, pp. 103-112 (1999); andcompounds that enhance the development of type 1 helper T cell response(Davis et al., “Future Options for the Management of Hepatitis C.”Seminars in Liver Disease, 19, pp. 103-112 (1999)). Also included arecompounds that stimulate the synthesis of interferon in cells(Tazulakhova, E. B. et al., “Russian Experience in Screening, analysis,and Clinical Application of Novel Interferon Inducers” J. InterferonCytokine Res., 21 pp. 65-73) including, but are not limited to, doublestranded RNA, alone or in combination with tobramycin, and Imiquimod (3MPharmaceuticals; Sauder, D. N. “Immunomodulatory and PharmacologicProperties of Imiquimod” J. Am. Acad. Dermatol., 43 pp. S6-11 (2000).

Compounds that stimulate the synthesis of interferon in cells(Tazulakhova, E. B. et al., “Russian Experience in Screening, analysis,and Clinical Application of Novel Interferon Inducers” J. InterferonCytokine Res., 21 pp. 65-73) include, but are not limited to, doublestranded RNA, alone or in combination with tobramycin, and Imiquimod (3MPharmaceuticals; Sauder, D. N. “Immunomodulatory and PharmacologicProperties of Imiquimod” J. Am. Acad. Dermatol., 43 pp. S6-11 (2000).

Other non-immunomodulatory or immunomodulatory compounds may be used incombination with a compound of this invention including, but not limitedto, those specified in WO 02/18369, which is incorporated herein byreference (see, e.g., page 273, lines 9-22 and page 274, line 4 to page276, line 11).

Still other agents include those described in various published U.S.Patent Applications. These publications provide additional teachings ofcompounds and methods that could be used in combination with VX-950 inthe methods of this invention, particularly for the treatment ofhepatitis. It is contemplated that any such methods and compositions maybe used in combination with the methods and compositions of the presentinvention. For brevity, the disclosure the disclosures from thosepublications is referred to be reference to the publication number butit should be noted that the disclosure of the compounds in particular isspecifically incorporated herein by reference. Exemplary suchpublications include U.S. Patent Publication No. 20040058982; U.S.Patent Publication No. 20050192212; U.S. Patent Publication No.20050080005; U.S. Patent Publication No. 20050062522; U.S. PatentPublication No. 20050020503; U.S. Patent Publication No. 20040229818;U.S. Patent Publication No. 20040229817; U.S. Patent Publication No.20040224900; U.S. Patent Publication No. 20040186125; U.S. PatentPublication No. 20040171626; U.S. Patent Publication No. 20040110747;U.S. Patent Publication No. 20040072788; U.S. Patent Publication No.20040067901; U.S. Patent Publication No. 20030191067; U.S. PatentPublication No. 20030187018; U.S. Patent Publication No. 20030186895;U.S. Patent Publication No. 20030181363; U.S. Patent Publication No.20020147160; U.S. Patent Publication No. 20040082574; U.S. PatentPublication No. 20050192212; U.S. Patent Publication No. 20050187192;U.S. Patent Publication No. 20050187165; U.S. Patent Publication No.20050049220; and U.S. Patent Publication No. US2005/0222236.

This invention may also involve administering a cytochrome P450monooxygenase inhibitor. CYP inhibitors may be useful in increasingliver concentrations and/or increasing blood levels of compounds thatare inhibited by CYP.

If an embodiment of this invention involves a CYP inhibitor, any CYPinhibitor that improves the pharmacokinetics of the relevant NS3/4Aprotease may be used in a method of this invention. These CYP inhibitorsinclude, but are not limited to, ritonavir (WO 94/14436), ketoconazole,troleandomycin, 4-methyl pyrazole, cyclosporin, clomethiazole,cimetidine, itraconazole, fluconazole, miconazole, fluvoxamine,fluoxetine, nefazodone, sertraline, indinavir, nelfinavir, amprenavir,fosamprenavir, saquinavir, lopinavir, delavirdine, erythromycin, VX-944,and VX-497. Preferred CYP inhibitors include ritonavir, ketoconazole,troleandomycin, 4-methyl pyrazole, cyclosporin, and clomethiazole. Forpreferred dosage forms of ritonavir, see U.S. Pat. No. 6,037,157, andthe documents cited therein: U.S. Pat. No. 5,484,801, U.S. applicationSer. No. 08/402,690, WO 95/07696 and WO 95/09614.

Methods for measuring the ability of a compound to inhibit cytochromeP450 monooxygenase activity are known. See, e.g., U.S. Pat. No.6,037,157, and Yun, et al. Drug Metabolism & Disposition, vol. 21, pp.403-407 (1993).

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease.Patients may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of active ingredients will also depend upon the particulardescribed compound and the presence or absence and the nature of theadditional anti-viral agent in the composition.

According to another embodiment, the invention provides a method fortreating a patient infected with a virus characterized by a virallyencoded serine protease that is necessary for the life cycle of thevirus by administering to said patient a pharmaceutically acceptablecomposition of this invention. In one embodiment, the methods of thisinvention are used to treat a patient suffering from a HCV infection.Such treatment may completely eradicate the viral infection or reducethe severity thereof. In another embodiment, the patient is a humanbeing.

In an alternate embodiment, the methods of this invention additionallycomprise the step of administering to said patient an anti-viral agentpreferably an anti-HCV agent. Such anti-viral agents include, but arenot limited to, immunomodulatory agents, such as α-, β-, andγ-interferons, pegylated derivatized interferon-α compounds, andthymosin; other anti-viral agents, such as ribavirin, amantadine, andtelbivudine; other inhibitors of hepatitis C proteases (NS2-NS3inhibitors and NS3-NS4A inhibitors); inhibitors of other targets in theHCV life cycle, including but not limited to helicase and polymeraseinhibitors; inhibitors of internal ribosome entry; broad-spectrum viralinhibitors, such as IMPDH inhibitors (e.g., VX-497 and other IMPDHinhibitors disclosed in U.S. Pat. Nos. 5,807,876 and 6,498,178,mycophenolic acid and derivatives thereof); inhibitors of cytochromeP-450, such as ritonavir, or combinations of any of the above.

Such additional agent may be administered to said patient as part of asingle dosage form comprising both a compound of this invention and anadditional anti-viral agent. Alternatively the additional agent may beadministered separately from the compound of this invention, as part ofa multiple dosage form, wherein said additional agent is administeredprior to, together with or following a composition comprising a compoundof this invention.

Pharmaceutical compositions may also be prescribed to the patient in“patient packs” containing the whole course of treatment in a singlepackage, usually a blister pack. Patient packs have an advantage overtraditional prescriptions, where a pharmacist divides a patients supplyof a pharmaceutical from a bulk supply, in that the patient always hasaccess to the package insert contained in the patient pack, normallymissing in traditional prescriptions. The inclusion of a package inserthas been shown to improve patient compliance with the physician'sinstructions.

It will be understood that the administration of the combination of theinvention by means of a single patient pack, or patient packs of eachformulation, containing within a package insert instructing the patientto the correct use of the invention is a desirable additional feature ofthis invention.

According to a further aspect of the invention is a pack comprising atleast one compound of formula I (in dosages according to this invention)and an information insert containing directions on the use of thecombination of the invention. Any composition, dosage form, therapeuticregimen or other embodiment of this invention may be presented in apharmaceutical pack. In an alternative embodiment of this invention, thepharmaceutical pack further comprises one or more of additional agent asdescribed herein. The additional agent or agents may be provided in thesame pack or in separate packs.

Another aspect of this involves a packaged kit for a patient to use inthe treatment of HCV infection or in the prevention of HCV infection (orfor use in another method of this invention), comprising: a single or aplurality of pharmaceutical formulation of each pharmaceuticalcomponent; a container housing the pharmaceutical formulation(s) duringstorage and prior to administration; and instructions for carrying outdrug administration in a manner effective to treat or prevent HCVinfection.

Accordingly, this invention provides kits for the simultaneous orsequential administration of a dose of at least one compound of formulaI (and optionally an additional agent). Typically, such a kit willcomprise, e.g. a composition of each compound and optional additionalagent(s) in a pharmaceutically acceptable carrier (and in one or in aplurality of pharmaceutical formulations) and written instructions forthe simultaneous or sequential administration.

In another embodiment, a packaged kit is provided that contains one ormore dosage forms for self administration; a container means, preferablysealed, for housing the dosage forms during storage and prior to use;and instructions for a patient to carry out drug administration. Theinstructions will typically be written instructions on a package insert,a label, and/or on other components of the kit, and the dosage form orforms are as described herein. Each dosage form may be individuallyhoused, as in a sheet of a metal foil-plastic laminate with each dosageform isolated from the others in individual cells or bubbles, or thedosage forms may be housed in a single container, as in a plasticbottle. The present kits will also typically include means for packagingthe individual kit components, i.e., the dosage forms, the containermeans, and the written instructions for use. Such packaging means maytake the form of a cardboard or paper box, a plastic or foil pouch, etc.

A kit according to this invention could embody any aspect of thisinvention such as any composition, dosage form, therapeutic regimen, orpharmaceutical pack. The packs and kits according to this inventionoptionally comprise a plurality of compositions or dosage forms.Accordingly, included within this invention would be packs and kitscontaining one composition or more than one composition.

In yet another embodiment the present invention provides a method ofpre-treating a biological substance intended for administration to apatient comprising the step of contacting said biological substance witha pharmaceutically acceptable composition comprising a compound of thisinvention. Such biological substances include, but are not limited to,blood and components thereof such as plasma, platelets, subpopulationsof blood cells and the like; organs such as kidney, liver, heart, lung,etc; sperm and ova; bone marrow and components thereof, and other fluidsto be infused into a patient such as saline, dextrose, etc.

According to another embodiment the invention provides methods oftreating materials that may potentially come into contact with a viruscharacterized by a virally encoded serine protease necessary for itslife cycle. This method comprises the step of contacting said materialwith a compound according to the invention. Such materials include, butare not limited to, surgical instruments and garments (e.g. clothes,gloves, aprons, gowns, masks, eyeglasses, footwear, etc.); laboratoryinstruments and garments (e.g. clothes, gloves, aprons, gowns, masks,eyeglasses, footwear, etc.); blood collection apparatuses and materials;and invasive devices, such as, for example, shunts and stents.

In another embodiment, the compounds of this invention may be used aslaboratory tools to aid in the isolation of a virally encoded serineprotease. This method comprises the steps of providing a compound ofthis invention attached to a solid support; contacting said solidsupport with a sample containing a viral serine protease underconditions that cause said protease to bind to said solid support; andeluting said serine protease from said solid support. In one embodiment,the viral serine protease isolated by this method is HCV NS3-NS4Aprotease.

All references cited within this document are incorporated herein byreference.

IV. Methods and Examples

In order that the invention described herein may be more fullyunderstood, the following methods and examples are provided. It shouldbe understood that these methods and examples are for illustrativepurposes only and are not to be construed as limiting this invention inany manner.

A. Preparation of Intermediates for Compounds of Formula I

Set forth below are various methods for preparing intermediates that canbe used to synthesize the compound of Formula I.

Preparation of 3-(benzyloxycarbonylamino)-4-cyclobutyl-2-hydroxybutanoicacid

A solution of the cyanohydrin prepared according to methods described inWO 04/113294 (1 g, 3.65 mmol) in conc. HCl (12 mL) was heated to refluxfor 18 hours. The reaction was concentrated in vacuo to afford thedesired amino acid as an HCl salt (1.7 g) which was used in the nextstep without further purification. A solution of the above HCl salt inTHF was treated with DIPEA (2.68 g) and Z—OSu (5.16 g). The reactionmixture was stirred at room temperature for 8 hours. The reactionmixture was diluted with toluene and HCl (12 N, until pH=1). Afterseparation, the organic layer was extracted with sat. NaHCO₃ (50 mL,twice). The aqueous layer was made acidic with HCl (6 N) until pH=1 andextracted with EtOAc (200 mL). The combined organic layer was dried andconcentrated in vacuo to afford the title compound (0.6 g). (M+1) 308.

Preparation of benzyl1-cyclobutyl-3-hydroxy-4-(methylamino)-4-oxobutan-2-ylcarbamate

To a solution of3-(benzyloxycarbonylamino)-4-cyclobutyl-2-hydroxybutanoic acid (250 mg,0.81 mmol) in DCM (20 mL) was added HOSu (140 mg, 1.22 mmol), EDC (234mg, 1.22 mmol). After stiffing for 1 hour, methylamine in THF (2 N, 0.81mL) was added to the above mixture. The reaction mixture was stirred for18 hours and then concentrated in vacuo. The residue was purified byGilson Prep to afford the title compound (135 mg). ¹H-NMR (CDCl₃): δ7.54-7.28 (m, 5H), 6.67 (NH, 1H), 5.03 (dd, 2H), 3.68 (m, 1H), 2.73 (m,3H), 2.26 (m, 1H), 1.97-1.31 (m, 9H). (M+1) 321.

Preparation of benzyl1-cyclobutyl-4-(cyclopropylamino)-3-hydroxy-4-oxobutan-2-ylcarbamate

To a solution of3-(benzyloxycarbonylamino)-4-cyclobutyl-2-hydroxybutanoic acid (600 mg,1.95 mmol) in DCM (20 mL) was added HOSu (337 mg, 2.93 mmol), EDC (562mg, 2.93 mmol). After stiffing for 1 hour, cyclopropylamine (223 mg, 3.9mmol) was added to the above mixture. The product was extracted withEtOAc. The combined organic layer was then washed with HCl (1N), water,NaHCO₃, and brine and then concentrated in vacuo to afford benzyl1-cyclobutyl-4-(cyclopropylamino)-3-hydroxy-4-oxobutan-2-ylcarbamate(530 mg). (M+1) 347.

Preparation of 3-amino-4-cyclobutyl-N-cyclopropyl-2-hydroxybutanamide

To a solution of the CBz amide (530 mg, 1.53 mmol) in MeOH (30 mL) wasadded Pd(OH)₂/C (106 mg). The mixture was stirred under H₂ (1 atm) for18 hours. After filtration, the filtrate was concentrated in vacuo toafford the title compound (300 mg). ¹H-NMR (CDCl₃): δ 3.29 (m, 1H), 2.74(m, 1H), 2.37-1.66 (m, 9H), 1.40 (m, 1H), 0.78 (m, 2H), 0.51 (m, 2H).(M+1) 213.

The following compounds were prepared in a similar fashion to preparing3-amino-4-cyclobutyl-N-cyclopropyl-2-hydroxybutanamide by using theappropriate amine:

Preparation of 3-amino-N-cyclopropyl-2-hydroxyhept-6-ynamide

3-Amino-N-cyclopropyl-2-hydroxyhept-6-ynamide was prepared as describedby N. Kobayashi, et al. in US 2003/153788, which is incorporated hereinby reference in its entirety. ¹H-NMR (500 MHz, DMSO-d₆): 8.18 (s), 6.34(s), 4.22 (s), 3.45 (s), 3.17 (s), 2.84 (s), 2.69 (d, J=3.2 Hz), 2.30(m), 2.24 (m), 1.70 (m), 1.59 (m), 0.62 (d, J=5.0 Hz), 0.53 (s) ppm; FIAm/z 197.01 ES⁺.

Preparation of Cbz-protected(3S)-3-amino-4-cyclopropyl-2-hydroxy-N-methylbutanamide

Step 1: Preparation ofbenzyl(2S)-1-cyano-3-cyclopropyl-1-hydroxypropan-2-ylcarbamate

To a solution of the aldehyde (7.9 g, 32 mmol) in MeOH (50 mL) at 10° C.was added Na₂S₂O₄ (6.13 g, 35.2 mmol) and the resulting mixture waswarmed to room temperature and stirred for 2 hours then cooled to 10° C.To this reaction mixture, a solution of KCN in water (50 mL) was added.After stirring at room temperature for 18 hours, the mixture wasextracted with TBME (100 mL, twice). The combined organic layers werewashed with water and brine, dried and concentrated in vacuo to affordthe title compound (8 g). (M+1) 275.

Step 2: Preparation of (3S)-methyl3-(benzyloxycarbonylamino)-4-cyclopropyl-2-hydroxybutanoate

To a solution of the cyanohydrin (1 g, 3.65 mmol) in MeOH (15 mL) at−20° C. was bubbled through a stream of dry HCl gas for 30 minutes. Theresulting mixture was stirred at room temperature for 2 hours. Thereaction mixture was purged with nitrogen gas for 30 minutes and thenconcentrated. The residue at 0° C. was quenched with ice water and thenstirred at room temperature for 1 hour. The product was extracted withEtOAc. The combined organic layer was washed with NaHCO₃, water, brineand concentrated in vacuo to afford the title compound (0.5 g). ¹H-NMR(CDCl₃) δ: 7.31-7.30 (m, 5H), 5.09 (d, 2H), 4.44-4.14 (m, 2H), 3.78 (d,3H), 1.58-1.42 (m, 2H), 0.70 (m, 1H), 0.47 (t, 2H), 0.11-0.01 (m, 2H).(M+1) 308.

Step 3: Preparation of(3S)-3-(benzyloxycarbonylamino)-4-cyclopropyl-2-hydroxybutanoic acid

To a solution of the methyl ester of Step 2 (400 mg; 1.3 mmol) in THF (8mL) and water (6.63 mL) was added LiOH (1 N; 1.37 mL). The reactionmixture was stirred for 30 minutes and then acidified with 1.0 N HCl topH=3˜4. The mixture was extracted with EtOAc (20 mL, twice). Thecombined organic layer was washed with water, brine, and thenconcentrated in vacuo to afford the title compound (370 mg). (M+1) 294.

Step 4: Preparation ofbenzyl(2S)-1-cyclopropyl-3-hydroxy-4-(methylamino)-4-oxobutan-2-ylcarbamate

To a solution of(3S)-3-(benzyloxycarbonylamino)-4-cyclopropyl-2-hydroxybutanoic acid(180 mg, 0.26 mmol) in DCM (20 mL) was added HOSu (105 mg, 0.92 mmol),EDC (175 mg, 0.92 mmol). After stirred for 30 minutes, methylamine inTHF (2 N, 0.92 mL) was added to above mixture. The reaction mixture wasstirred for 18 hours and then concentrated in vacuo. The residue waspurified by Gilson Prep to afford title compound (50 mg). ¹H-NMR(CDCl₃): δ 7.53-7.26 (m, 5H), 6.83 (NH, 1H), 5.25 (NH, 1H), 5.05 (m,2H), 4.25-3.89 (m, 3H), 2.70 (m, 3H), 1.4 (m, 1H), 0.86 (m, 1H), 0.61(m, 1H), 0.38 (m, 2H), 0.33 (m, 2H). (M+1) 307.

The following compounds can be prepared in the similar manner by usingappropriate amines, followed by hydrogenation.

The following compounds can be prepared in the methods described byPerni, R. et al. in WO 01/74768, which is incorporated herein byreference in its entirety.

Preparation of (S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoicacid

In a 5 L RB flask dissolved t-butyl glycine (74 g, 0.56 mol, 1.02 eq.)in saturated sodium bicarbonate (11 vol). Cyclopentyl2,5-dioxopyrrolidin-1-yl carbonate (126 g, 0.55 mol, 1 eq.) wasdissolved in acetone (5.5 vol) and the solution slowly added viaaddition funnel at room temperature to the solution of the glycine. Thereaction mixture was stirred at room temperature until complete(approximately 4 hours). The acetone was removed under reduced pressureand the remaining aqueous solution was extracted with 30% ethyl acetatein hexanes (thrice, 5.5 vol each). The organic layers were discarded.The pH of the aqueous layer was adjusted to 2 with 2 N HCl and thenextracted with ethyl acetate (thrice, 5.5 vol). The combined organiclayers were dried (Na₂SO₄), filtered, and the solvent removed underreduced pressure to provide a clear oil the slowly crystallized. Thecrude product was crystallized from hexanes/ethyl acetate to provide(S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoic acid as a whitesolid (82 g). The mother liquid was stripped and a second crop ofcrystals obtained (combined yield 105.54 g, 79% yield).

Preparation of Sulfonyl Compounds

Compounds S1, S2, S3, and S4, shown above, were prepared according toprocedures described in WO 2005/095403 and PCT/US2005/010494, herebyincorporated by references by their entireties. Specifically, to asolution of chlorosulfonylisocyanate (10 mL, 115 mmol) in CH₂Cl₂ (200mL) at 0° C. was added t-BuOH (11 mL, 1 eq.). The mixture was stirredfor 60 minutes, then added via cannula into a solution ofcyclopropylamine (6.6 g) in CH₂Cl₂ (200 mL) with triethylamine (30 mL)at 0° C. concurrently with a solution of triethylamine (50 mL) in CH₂Cl₂(100 mL) via addition funnel. Internal temperature was maintained below8° C. Stirred at room temperature after completion of addition for 4hours. The reaction was then diluted with CH₂Cl₂ and transferred to aseparatory funnel, washed with 1 N HCl (twice, 400 mL each), brine (300mL), dried (MgSO₄), filtered and concentrated. The product wasrecrystallized from ethyl acetate/hexanes to yield 16.8 g (71.3 mmol,62%) of S3. Compound S3 was deprotected with trifluoroacetic acid inCH₂Cl₂ to give compound S4 in quantitative yield.

Ammonia gas was bubbled through a gas dispersion tube into THF (40 mL)cooled to 0° C. for 5 minutes. To this solution at 0° C. was addedcyclopropylsulfonylchloride (1 gram, 7.1 mmol). The reaction was stirredat room temperature overnight, then filtered through a plug of silicagel, followed by elution with EtOAc to yield 750 mg (6.19 mmol, 87%) ofcyclopropylsulfonamide. ¹H-NMR (500 MHz, Methanol-d₄): 4.79 (s, 2H),2.59-2.54 (m, 1H), 1.06-0.96 (m, 4H).

To a solution of compound XX5 (1.37 g, 6.41 mmol) in THF (30 mL) at 0°C. was added dropwise borane-dimethylsulfide (3.85 mL, 7.8 mmol, 2.0 Min toluene). The reaction mixture was stirred for 1 h with gradualwarming to room temperature, quenched with H₂O (20 mL), and extractedwith ethyl acetate (thrice, 30 mL each). The combined organics weredried and concentrated under reduced pressure to provide 1.3 g of acolorless oil which was used without further purification. To oxalylchloride (2.24 mL, 25.6 mmol) in CH₂Cl₂ (15 mL, anhydrous) at −78° C.under inert atmosphere was added dropwise a solution of DMSO (2.73 mL,38.5 mmol) in CH₂Cl₂ (8 mL). After stirring for 10 min, a solution ofthe alcohol (1.3 g, 6.41 mmol) in CH₂Cl₂ (6 mL) was added dropwise.After an additional 10 min, triethylamine (7.15 mL, 51.3 mmol) in CH₂Cl₂was added and the reaction was stirred another 30 min with gradualwarming to 0° C. The reaction mixture was washed with 1 M HCl (20 mL)followed by brine (20 mL). The organic layer was dried over MgSO₄ andconcentrated under reduced pressure. The resulting oil was purified viasilica gel chromatography to afford 748 mg (59% over 2 steps) ofaldehyde XX6. ¹H-NMR (500 MHz, CDCl3): 9.75 (s, 1H), 3.67 (s, 3H),2.91-2.85 (m, 1H), 2.78-2.74 (m, 1H), 2.56-2.52 (m, 1H), 1.74-1.71 (m,2H), 1.66-1.58 (m, 4H), 1.27-0.95 (m, 5H).

To a solution of compound XX6 (581 mg, 2.9 mmol) and K₂CO₃ (811 mg, 5.9mmol) in MeOH (15 mL) was added dimethyl 1-diazo-2-oxopropylphosphonate(676 mg, 3.5 mmol, Synlett 1996, p. 521). The reaction was stirred 1 hat room temperature, diluted with Et₂O (20 mL), and washed withsaturated NaHCO₃ solution (10 mL, aqueous). The organic layer was driedover MgSO₄ and concentrated under reduced pressure to give 600 mg (100%)of alkyne XX7 which was used without further purification. ¹H-NMR (500MHz, CDCl₃): 3.69 (s, 3H), 2.48-2.37 (m), 1.95 (s, H), 1.73-1.60 (m),1.30-0.94 (m).

To a solution of compound XX7 (600 mg, 2.9 mmol) in a solution ofTHF/H₂O/MeOH (25 mL, 2:1:2) was added LiOH monohydrate (850 mg, 20.3mmol). The reaction mixture was stirred 2 h at room temperature,acidified using 1 N HCl (25 mL), and extracted with EtOAc (thrice, 15 mLeach). The combined organics were dried over MgSO₄ and concentrated toyield 533 mg (99%) of carboxylic acid XX8, which was used withoutfurther purification.

To a solution of compound XX5 (100 mg, 0.5 mmol) in CH₂Cl₂ (2.5 mL) wasadded EDC (107 mg, 0.6 mmol), HOBt (76 mg, 0.6 mmol) and triethylamine(195 μL, 1.4 mmol). To the activated acid solution was added methylaminehydrochloride (38 mg, 0.6 mmol) and the reaction was stirred at roomtemperature for 12 h. The reaction mixture was washed with H₂O (2 mL), 1N HCl (2 mL) and saturated NaHCO₃ solution (2 mL). The organic layer wasdried over MgSO₄ and concentrated to give 100 mg of amide XX9, which wasused without further purification. ¹H-NMR (500 MHz, CDCl3) 3.61 (s, 3H),2.75-2.70 (m, 4H), 2.48-2.42 (m, 1H), 2.28-2.24 (m, 1H), 1.66-1.48 (m,6H), 1.35-0.90 (m, 5H).

To a solution of compound XX9 (100 mg, 0.5 mmol) in a solution ofTHF/H₂O/MeOH (3 mL, 2:1:2) was added LiOH monohydrate (124 mg, 3 mmol).The reaction mixture was stirred 2 h at room temperature, acidifiedusing 1 N HCl (4 mL), and extracted with EtOAc (3×5 mL). The combinedorganics were dried over MgSO₄ and concentrated to yield 87 mg ofcarboxylic acid XX10, which was used without further purification.¹H-NMR (500 MHz, CDCl3) 11.32 (s, H), 2.75-2.64 (m, H), 2.52-2.46 (m,H), 2.37-2.33 (m, H), 2.25 (td, J=8.7, 2.9 Hz, H), 1.97 (s, H), 1.79 (s,H), 1.74-1.62 (m, H), 1.59-1.49 (m, H), 1.23-1.12 (m, H), 1.08-0.81 (m,H).

Intermediate XX12 was prepared according to the procedure for preparingintermediate XX10 described above, except for using pyrrolidine as areagent instead of methylamine hydrochloride. ¹H-NMR (500 MHz, CDCl3)11.47 (s, 1H), 3.45-3.32 (m, 4H), 2.76-2.72 (m, 1H), 2.64-2.59 (m, 1H),2.37-2.33 (m, 1H), 1.92-1.76 (m, 4H), 1.71-1.57 (m), 1.22-0.84 (m).

To a solution of compound XX5 (1 g, 4.7 mmol) and HgO yellow (1.01 g,4.7 mmol) in CCl₄ (23 mL) at reflux was added dropwise over 30 min asolution of bromine (264 μL, 5.1 mmol) in CCl₄ (5 mL). The reaction wasstirred at reflux for 1 h, cooled to room temperature, diluted withCH₂Cl₂ (20 mL), washed with 1 N HCl (10 mL), H₂O (10 mL), and brine (10mL). The organic layer was dried over MgSO₄ and concentrated underreduced pressure to yield 1.3 g of compound XX13 as a colorless oil thatwas used without further purification. ¹H-NMR (500 MHz, CDCl₃): 3.67 (s,3H), 3.52-3.44 (m, 2H), 2.63-2.58 (m, 1H), 1.70-1.64 (m, 3H), 1.60-1.54(m, 3H), 1.24-0.92 (m, 5H).

To a solution of compound XX13 (578 mg, 2.3 mmol) in DMSO (12 mL) wasadded sodium borohydride (177 mg, 4.7 mmol). The reaction mixture wasstirred at 90° C. for 1 h, diluted with H₂O (10 mL), and extracted withhexanes (3×15 mL). The combined organics were dried over MgSO₄ andconcentrated under reduced pressure. Purification via silica gelchromatography, eluting with EtOAc/petroleum ether, afforded 204 mg ofcompound XX14. ¹H-NMR (500 MHz, CDCl₃): 3.59 (s, 3H), 2.18 (m, 1H),1.69-1.43 (m, 6H), 1.21-0.83 (m, 8H).

Intermediate XX15 was prepared according to the procedure for preparingintermediate XX10, step b, except for using substrate XX14 instead ofXX9.

To a solution of (S)-2-amino-3,3-dimethylbutanoic acid (787 mg, 6.0mmol), bromobenzene (632 μL, 6.0 mmol), K₂CO₃ (1.24 g, 9.0 mmol) and CuI(114 mg, 0.6 mmol) was added N,N-dimethylacetamide (7.5 mL). Thecontents were stirred for 16 h at 90° C. in a sealed pressure vessel.The reaction mixture was diluted with H₂O (15 mL), cooled to 0° C., andacidified to pH˜5 using 1 N HCl. The mixture was extracted with EtOAc(3×20 mL), and the combined organics were washed with brine (1×15 mL),dried over MgSO₄, and concentrated under reduced pressure. The resultingresidue was purified via silica gel chromatography to provide 150 mg(12%) of compound XX16. ¹H-NMR (500 MHz, CDCl3): 7.11-7.09 (m, 2H), 6.69(t, J=7.3 Hz, 1H), 6.60-6.59 (m, 2H), 3.69 (s, 1H), 1.02 (s, 9H).

Intermediate XX17 was prepared according to the procedure for preparingXX16, except for using 1-bromo-3-methoxybenzene as a reagent instead ofbromobenzene. ¹H-NMR (500 MHz, CDCl₃): 6.98 (t, J=8.1 Hz, 1H), 6.24-6.18(m, 2H), 6.14 (s, 1H), 3.69 (s, 1H), 3.66 (s, 3H), 1.00 (s, 9H).

To a solution of (S)-3-(methoxycarbonyl)-4-methylpentanoic acid (200 mg,1.2 mmol) in CH₂Cl₂ (6 mL) was added EDC (264 mg, 1.4 mmol), HOBt (186mg, 1.4 mmol) and triethylamine (481 μL, 3.5 mmol). To the activatedacid solution was added cyclohexylamine (158 μL, 1.4 mmol) and thereaction was stirred 4 hours. The reaction mixture was washed with H₂O(3 mL), 1 N HCl (3 mL), and saturated NaHCO₃ solution (3 mL). Theorganic layer was dried over MgSO₄, and concentrated under reducedpressure to afford 290 mg of compound XX18 which was used withoutfurther purification. ¹H-NMR (500 MHz, CDCl₃): 5.78 (d, J=7.5 Hz, 1H),3.69-3.61 (m, 4H), 2.73-2.69 (m, 1H), 2.45-2.40 (m, 1H), 2.24-2.20 (m,1H), 1.85 (m, 1H), 1.82-1.76 (m, 2H), 1.63-1.60 (m, 2H), 1.54-1.50 (m,1H), 1.31-1.22 (m, 2H), 1.12-1.00 (m, 3H), 0.90-0.85 (m, 6H).

Intermediate XX19 was prepared according to the procedure for preparingcompound XX10 described above, except for using substrate XX18 as areagent instead of compound XX9. ES (+) MS: m/e 256 (M+H)⁺.

Intermediate XX20 was prepared according to the procedure for preparingcompound XX18 or XX19 described above, except for using isopropylamineas a reagent instead of cyclohexylamine. ES (+) MS: m/e 216 (M+H)⁺.

Intermediate XX21 was prepared according to the procedure for preparingXX18 or XX19 described above, except for using benzylamine as a reagentinstead of cyclohexylamine. ES (+) MS: m/e 264 (M+H)⁺.

Glycine methyl ester hydrochloride (50.0 g) was suspended in MTBE (300mL) at RT. To this was added benzaldehyde (40.5 mL) and anhydrous Na2SO4(33.9 g). The suspension was cooled in an ice-water bath for 20 minutes,then triethylamine (80 mL) was added dropwise over 15 minutes. After 5minutes, the reaction was removed from the ice-water bath, and stirredat RT for 24 hours. The reaction was quenched with 200 mL ice-watermixture and the organic layer was separated. The aqueous layer wasextracted with MTBE (200 mL). The organic layers were combined, washedwith a 1:1 mixture of brine and saturated NaHCO3 (aq.), dried (MgSO₄),and concentrated to yield 62.83 grams of the N-benzyl imine as a yellowoil. ¹H-NMR (500 MHz, CDCl₃): 8.30 (s, 1H), 7.78-7.77 (m, 2H), 7.45-7.40(m, 3H), 4.42 (s, 2H), 3.78 (s, 3H).

Lithium tert-butoxide (15.13 g) was suspended in dry toluene (200 mL) atroom temperature. To this was added dropwise a solution of the N-benzylimine of glycine methyl ester (16.89 g) and 1,4-dibromo-2-butene (19.28g) in toluene (100 mL) over 40 minutes. The red solution was stirred for100 minutes, then quenched with H₂O (200 mL). The contents weretransferred to a separatory funnel and diluted with MTBE (200 mL). Thelayers were separated and the aqueous layer was extracted with MTBE. Thecombined organic layers were stirred with 1 N HCl (aq.) (500 mL) for 3hours. The layers were separated and the organic layer was extractedwith H₂O (100 mL). The aqueous layers were combined, NaCl (250 g) andMTBE (700 mL) were added and the pH was brought to ˜13 with 10 N NaOH(aq). The organic layer was separated and the aqueous layer wasextracted with MTBE (twice, 300 mL each). The organic layers werecombined, dried (MgSO₄), and concentrated to a volume of ˜400 mL. To thesolution was added di-tert-butyl dicarbonate (25.0 g) and the reactionwas stirred for 3 days. Additional di-tert-butyl dicarbonate (5.6 g) wasadded, followed by heating of the reaction in a 60° C. bath for 1 hour.The reaction was purified by flash silica gel column chromatography withEtOAc/hexane (1:9) as eluent to yield 10.89 g of racemicN-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid methylester. See, e.g., WO00/09558 and Beaulieu, P. L. et al., J. Org. Chem.,70 (15), 5869-5879, 2005. ¹H-NMR (500 MHz, CDCl₃): 5.78-5.71 (m, 1H),5.29-5.26 (m, 1H), 5.11 (dd, J=1.2, 10.3 Hz, 1H), 3.71 (s, 3H), 2.14 (q,J=8.8 Hz, 1H), 1.79 (s, 1H), 1.53-1.45 (m, 10H).

Racemic N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylicacid methyl ester (4.2 g) was dissolved in acetone (80 mL) and thendiluted with water (160 mL). The pH was adjusted to 7.8 with 0.2N NaOH(aq). Subtilisin A (product P-5380 from Sigma, St. Louis, Mo., USA) (4.5g) was added to the solution. Its pH was maintained between 7.4 and 8.7for 3 days by the dropwise addition of 0.1 N NaOH (aq.). When HPLCanalysis (Chiralpak AD from Daicel Chemical Industries, Tokyo, 4.6mm×250 mm, 0.5 mL/min, 10-85% 2-propanol/hexanes over 10 minutes,monitor 215.4 nm) of the reaction indicated the presence of only the(1R,2S)-enantiomer (retention time of (1R,2S)=6.2 min, (1S,2R)=5.9 min)the pH was brought to 8.5 with 2 N NaOH (aq). The contents of thereaction were transferred to a separatory funnel and extracted with MTBE(3×400 mL). The extracts were washed with saturated NaHCO3 (aq) solution(3×150 mL), water (2×200 mL), and dried (MgSO4). The solution wasfiltered, concentrated, diluted with CH2Cl2, dried (MgSO4), filtered,and concentrated to yield 1.95 g ofN-Boc-(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid methyl ester.

N-Boc-(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid methyl ester(125 mg, 0.52 mmol) stirred in CH2Cl2/TFA (1:1, 2 mL) at RT for 90minutes. Solvents removed under vacuum to yield(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid methyl estertrifluoroacetic acid salt.

Compound XX1 (2.34 g, 9.71 mmol) was stirred with LiOH.H₂O (0.45 g, 10.7mmol) in THF/H₂O/THF (3:1:0.5, 22 mL) at room temperature overnight. Thesolvents were evaporated and the remaining solids were taken up inCH₂Cl₂/EtOAc and 1N HCl (aq). The aqueous layer was extracted withCH₂Cl₂ and the combined organic extracts were dried (MgSO₄), filtered,and concentrated. This material was dissolved in CH₂Cl₂ (10 mL) at roomtemperature and treated with trifluoroacetic acid (10 mL). HPLC analysisat 70 minutes showed no starting material was present. The solvents wereremoved in vacuo to yield a viscous light colored oil. This was taken upin additional CH2Cl2 (30 mL) and evaporated on a rotary evaporator toyield a tan solid. This solid was dissolved in saturated NaHCO3 (aq) andacetone (1:1, 50 mL) and treated with Fmoc-Cl (2.65 g, 10.2 mmol). After4 hours, the contents of the flask were transferred to a separatoryfunnel with CH₂Cl₂ and acidified with 2N HCl (aq). The aqueous layer wasextracted with CH₂Cl₂, the combined organic layers were dried (MgSO4),filtered, and concentrated to yield 1.86 g (5.3 mmol) of XX2 as a lightyellow solid. (M+1)=350.1

PS-Wang resin (2.0 g, 1.0 eq.) swelled in DMF (enough to cover).(1R,2S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-vinylcyclopropanecarboxylicacid (XX3) (922 mg, 1.1 eq.) was stirred in DCM. Diisopropylcarbodiimide(409 uL, 1.1 eq.) was added to the DCM solution and stirred at 4° C. for2 hours, then added to resin and DMF. Dimethylaminopyridine (29 mg, 0.1eq.) in DMF was added to resin solution and shaken for 5 hours. Drainedand washed with DMF (thrice) and DCM (thrice) to yield Compound XX4.

Preparation of 2-(bicyclo[4.1.0]heptan-1-yl)acetic acid X2

Commercially available compound X1 (Aldrich Chemical Co., Milwaukee,Wis., USA) was converted to X2 according to method described by E. J.Kantorowski et al. in J. Org Chem., 1999, 64, 570-580. ¹H-NMR (CDCl₃,500 MHz): 9.2 (br s, 1H), 2.23 (m, 2H), 1.92 (m, 1H), 1.76 (m, 2H), 1.58(m, 1H), 1.34 (m, 1H), 1.18 (m, 4H), 0.85 (m, 1H), 0.52 (dd, 1H), 0.31(t, 1H) ppm.

Preparation of 2-(1-hydroxycyclohexyl)acetic acid X5

Compound X4 was prepared using essentially the procedure described inBull. Chem. Soc. Jpn., 1971, 44, 1090. Specifically, A solution ofethylbromoacetate (8.3 mL) (Aldrich Chemical Co., Milwaukee, Wis., USA)in toluene was added dropwise at 80° C. over 30 min to a thoroughlystirred mixture of cyclohexanone X3 (4.9 g) and zinc powder (4.9 g) intoluene. The addition was carefully monitored and the temperature waskept at 80° C. After the addition was completed, the mixture wasrefluxed for 90 min., cooled, decomposed with 1N aqueous HCl, andextracted with Et₂O. The organics were washed with water, aq. NaHCO₃,dried (MgSO₄) and concentrated in vacuo to yield X4 (5.9 g): ¹H-NMR(CDCl₃, 500 MHz) 4.16 (t, 2H), 3.0 (br s, 1H), 2.46 (s, 2H), 1.40-1.69(m, 10H), 1.27 (t, 3H) ppm; FIA m/z 187.1 ES⁺.

To a solution of X4 (510 mg) in MeOH was added 1N aqueous NaOH. Thereaction mixture was stirred at 60° C. for 1 h, and then concentrated invacuo. The residue was diluted with water, washed with Et₂O and theaqueous layer acidified with 1N aqueous citric acid and extracted withEtOAc. The organics were dried (MgSO₄) and concentrated in vacuo toyield after recrystallization compound X5 (220 mg): ¹H-NMR (CDCl₃, 500MHz) 3.63 (s, 1H), 2.45 (s, 2H), 1.22-1.64 (m, 10H) ppm; FIA m/z 157.2ES⁻.

Preparation of 2-(1-methylcyclohexyl)acetic acid (X8)

Commercially available compound X6 (Aldrich Chemical Co., Milwaukee,Wis., USA) was converted to compound X7 according to the methoddescribed by N. Asao et al. in Tetrahedron Lett., 2003, 44, 4265. ¹H-NMR(CDCl₃, 500 MHz): 4.12 (q, 2H), 2.22 (s, 2H), 1.30-1.48 (m, 10H), 1.25(t, 3H), 1.01 (s, 3H) ppm.

To a solution of compound X7 in EtOH was added 1 N aqueous NaOH. Thereaction mixture was stirred at 50° C. for 3 hours, and thenconcentrated in vacuo. The residue was diluted with water, washed withEt₂O and the aqueous layer acidified with 1 N aqueous citric acid andextracted with CH₂Cl₂. The organics were dried (MgSO₄) and concentratedin vacuo to yield compound X8. ¹H-NMR (CDCl₃, 500 MHz): 11.7 (s, 1H),2.26 (s, 2H), 1.32-1.49 (m, 10H), 1.05 (s, 3H) ppm.

Preparation of 2-(4-methyltetrahydro-2H-pyran-4-yl)acetic acid (X12)

To a solution of dihydro-2H-pyran-4(3H)-one (X9) (3.13 g, from Aldrich)in toluene was added (carbethoxymethylene)-triphenylphosphorane (12.0 g,Aldrich). The solution was stirred at 110° C. for 3 days. The resultingdark solution was concentrated in vacuo and the residue directlypurified by column over silica gel to yield compound X10 (4.54 g) as aclear liquid. ¹H-NMR (CDCl₃, 500 MHz): 5.66 (s, 1H), 4.16 (q, 2H), 3.98(s, 4H), 3.00 (t, 2H), 2.38 (m, 2H), 1.77 (m, 4H), 1.27 (t, 3H) ppm.

Compounds X11 and X12 were obtained in a similar manner as described forcompounds X7 and X8. ¹H-NMR (CDCl₃, 500 MHz): 3.64-3.73 (m, 4H), 2.35(s, 2H), 1.65 (ddd, 2H), 1.50 (ddt, 2H), 1.17 (s, 3H) ppm.

Preparation of 2-(cis-2,6-dimethyltetrahydro-2H-pyran-4-yl)acetic acid(X16)

Intermediate X13 was prepared from commercially available2,6-dimethyl-g-pyrone (Aldrich Chemical Co., Milwaukee, Wis., USA). Asolution of the g-pyrone was dissolved in EtOH and hydrogenated (2 atm.H₂) with 10% Pd/C over 2 h. The catalyst was subsequently filtered offand the solution was concentrated in vacuo to yield crude X13 which waspurified by column chromatography to yield pure compound X13. ¹H-NMR(CDCl₃, 500 MHz): 3.72 (m, 2H), 2.35 (m, 2H), 2.21 (dd, 2H), 1.32 (d,6H) ppm.

Compound X14 was then obtained from compound X13 in a similar manner asdescribed for compound X10. ¹H-NMR (CDCl₃, 500 MHz): 5.65 (s, 1H), 4.15(q, 2H), 3.80 (dt, 1H), 3.49 (m, 2H), 2.17 (dt, 1H), 2.07 (dd, 1H), 1.79(dt, 1H), 1.28 (m, 9H) ppm. LC-MS m/z 199.126 ES⁺.

A solution of compound X14 in EtOAc was then hydrogenated (1 atm. H₂)with 10% wet Pd/C over 1 hour. The catalyst was subsequently filteredoff and the solution was concentrated in vacuo to yield crude compoundX15 which was used without further purification for the next step.Compound X16 was then prepared from compound X15 in a similar manner asdescribed for compound X8. ¹H-NMR (CDCl₃, 500 MHz) major diastereomer:3.50 (m, 2H), 2.27 (d, 2H), 2.07 (m, 1H), 1.71 (m, 2H), 1.19 (d, 6H)0.92 (m, 2H) ppm; major diastereomer: 3.64 (m, 2H), 2.56 (d, 2H), 2.47(m, 1H), 1.49 (m, 2H), 1.15 (d, 6H), 0.86 (m, 2H) ppm.

Preparation of 2-(1,4-dioxaspiro[4.5]decan-8-yl)acetic acid X20

Compound X20 was prepared from compound X17 (from Aldrich) according tothe procedures described above for preparing compound X16.

Compound X18: ¹H-NMR (CDCl₃, 500 MHz): 5.66 (s, 1H), 4.15 (q, 2H), 3.98(s, 4H), 3.00 (m, 2H), 2.38 (m, 2H), 1.77 (m, 4H), 1.27 (t, 3H) ppm.

Compound X19: ¹H-NMR (CDCl₃, 500 MHz): 4.12 (q, 2H), 3.93 (s, 4H), (d,2H), 1.83 (m, 1H), 1.72 (m, 4H), 1.56 (dt, 2H), 1.33 (m, 2H), 1.30 (m,3H) ppm.

Compound X20: ¹H-NMR (CDCl₃, 500 MHz): 3.93 (s, 4H), 2.28 (d, 2H),1.73-1.86 (m, 4H), 1.57 (dt, 2H), 1.35 (m, 2H) ppm.

Preparation of 2-(trans-2,6-dimethyltetrahydro-2H-pyran-4-yl)acetic acid25

Compounds X21 and X22 were prepared according to the method described byS. Danishefsky et al. in J. Org. Chem. 1982, 47, 1597-1598 and D. S.Reddy et al. in J. Org. Chem. 2004, 69, 1716-1719, respectively.Compound X25 was prepared from compound X22 according to the methoddescribed above for preparing compound X16.

Compound X23. ¹H-NMR (CDCl₃, 500 MHz): 5.72 (s, 1H), 4.16 (q, 2H), 4.08(q, 2H), 3.06 (dd, 1H), 2.75 (dd, 1H), 2.39 (dd, 1H), 2.05 (dd, 1H),1.28 (t, 3H), 1.19 (m, 6H) ppm.

X25: ¹H-NMR (CDCl₃, 500 MHz) 4.24 (m, 1H), 3.78 (m, 1H), 2.25 (m, 3H),1.71 (m, 1H), 1.53 (m, 1H), 1.46 (m, 1H), 1.29 (d, 3H), 1.13 (d, 3H),0.90 (m, 1H) ppm.

Preparation of 2-(4-hydroxy-4-methylcyclohexyl)acetic acid X27

A solution of compound X20 in dioxane was treated with 4N HCl indioxane. The reaction solution was stirred at room temperature for 4hours and concentrated in vacuo to give crude compound X26 which wasused without further purification for the next step. To a stirredsolution of compound X26 in THF was slowly added MeMgBr (3 N in THF).The resulting mixture was stirred at 40° C. for 3 hours, quenched with 1N aqueous citric acid and diluted with EtOAc. The phase were separatedand the organics were dried (MgSO₄), concentrated in vacuo and purifiedby chromatography over silica gel to give compound X27 as a mixture oftwo diastereomers: isomer 1: ¹H-NMR (CDCl₃, 500 MHz): 4.50 (br s), 2.27(m, 2H), 1.75 (m, 1H), 1.65 (m, 4H), 1.39 (m, 4H), 1.22 (s, 3H) ppm;isomer 2: ¹H-NMR (CDCl₃, 500 MHz): 2.12 (m, 2H), 1.69 (m, 3H), 1.56 (m,2H), 1.39 (m, 2H), 1.12 (s, 3H), 1.05 (m, 2H) ppm.

Preparation of 2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)acetic acid

To a solution of the methyl ester (500 mg; 2.69 mmol) in THF (21.5 mL),MeOH (21.5 mL) and water (10.75 mL) was added LiOH (1 N; 10.75 mL). Thereaction mixture was stirred for 3 hours. The reaction was acidifiedwith HCl (1 N, pH=5). The product was extracted with EtOAc (twice, 20 mLeach). The combined organic layer was then wash with water, brine andconcentrated in vacuo to afford 420 mg of2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)acetic acid. ¹H-NMR (CDCl₃): δ3.76-3.67 (m, 2H), 2.56-2.19 (m, 3H), 1.63 (m, 2H), 1.26-1.10 (m, 8H).(M+1) 173.

To a solution of compound X30 (64 g, 237 mmol) and EDC (226 g, 1.19 mol)in EtOAc (1.5 L) was added DMSO (400 mL), and the resulting suspensionwas cooled to 0° C. To this mixture was added a solution ofdichloroacetic acid in EtOAc (1:1 v/v, 130 mL) keeping the internalreaction temperature below 25° C. The reaction was warmed to roomtemperature, stirred for 15 minutes, cooled to 0° C., and quenched with1 N HCl (1 L). The organic layer was separated, washed with H₂O (2×500mL), dried over MgSO₄, and concentrated under reduced pressure. Theresulting oil was filtered through a plug of silica eluting withEtOAc/hexanes to afford 48 g (76%) of compound X31 as a white solid.

To resin X32 (prepared according to the procedure described in WO00/23421) (100 g, 0.88 mmol/g) was added a solution of X31 (48 g, 179mmol) in THF (650 mL), followed by AcOH (30 mL). The mixture was shakenfor 16 hours, and the resin was filtered, washed with THF (4 times, 400mL each) and CH₂Cl₂ (4 times, 400 mL each) and dried in vacuo. Thefiltrate and washes were combined and concentrated, and the aboveprocedure was repeated to afford resin X33 with a loading ofapproximately 0.4 mmol/g.

Preparation of Aldehyde Compounds

5-chloronicotinaldehyde was prepared according to methods described byD. L. Comins et al. in Hetereocycles, 1987, 26 (8), pp. 2159-2164.

Some other aldehydes such as 2-fluoro-5-chlorobenzaldehyde,2-methoxy-3-methyl benzaldehyde, 2-methoxynicotinaldehyde,2,3-dihydrobenzofuran-7-carbaldehyde can be made from corresponding acidbased on following procedure:

Preparation of 2,3-dihydrobenzofuran-7-carbaldehyde

2,3-Dihydrobenzofuran-7-carboxylic acid (820 mg, 5 mmol) was dissolvedin THF (10 mL). To the solution was added TEA (0.7 mL, 5 mmol) andmethylchloroformate (0.43 mL, 5 mmol). The solution was stirred for 0.5hour. The white precipitates were removed by filtration, the filtratewas added to a solution of NaBH₄ (437 mg, 12.5 mmol) in H₂O (5 mL). Theresulting solution was stirred overnight. The reaction mixture wasneutralized with 2 M aqueous HCl solution and then extracted with EtOAc.The organic layer was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated in vacuo. The crude alcohol was dissolved in DCM. To thesolution was added PCC (1.83 g, 7.5 mmol). The mixture was stirred for 2hours at room temperature and diluted with diethyl ether, then etherlayers were decanted. Combined organic layer was filtered though a layerof Celite®. The filtrate was concentrated to give crude product. Thecrude was purified from column with 10% EtOAc/hexane to afford 450 mg of2,3-dihydrobenzofuran-7-carbaldehyde as a slightly yellow solid. HPLC4.3 min.

Preparation of 4-chloropicolinaldehyde

A suspension of MnO₂ (7.3 g, 84 mmol) and(4-chloro-pyrindin-2-yl)methanol (1 g, 7 mmol) in CHCl₃ was heated toreflux for 90 minutes. The mixture was filtered though a layer ofCelite® and concentrated in vacuo to afford 520 mg of4-chloropicolinaldehyde as a white solid. HPLC 1.8 minutes and MS 142 asM=1 peak.

Preparation of 3-chloro-5-methoxybenzaldehyde

A mixture of 3-chloro-5-methoxybenzyl alcohol (5.0 g, 28.9 mmol) andpyridinium chlorochromate (20% on alumina, 40 g, 37.8 mmol) was allowedto stir for 1.25 hr. Diethyl ether (200 ml) was then added followed byfiltration of precipitate. The filtrate was concentrated under reducedpressure and the resulting residue was purified via silica gelchromatography using 40% dichloromethane, 60% petroleum ether as eluant,to give 3.8 g of 3-chloro-5-methoxybenzaldehyde (78%). ¹H-NMR (CDCl₃):3.84 (s, 3H) 7.13 (s, 1H), 7.28 (s, 1H), 7.41 (s, 1H), 9.89 (s, 1H).

Preparation of 1-(bromomethyl)-3-chloro-5-methylbenzene

To a solution of m-chloroxylene (0.96 g, 6.8 mmol) in carbontetrachloride at reflux was added N-bromosuccinimide (1.4 g, 7.5 mmol)followed by benzoyl peroxide (1.6 g, 6.8 mmol). The reaction was allowedto stir for 20 minutes and cooled to room temperature, filtered offprecipitate and the filtrate was concentrated under reduced pressure andthe resulting residue was purified via silica gel chromatography usingpetroleum ether as eluant to give 0.89 g of1-(bromomethyl)-3-chloro-5-methylbenzene (60%). NMR (CDCl₃): 2.31 (s,3H) 4.37 (s, 2H) 7.09 (s, 1H) 7.12 (s, 1H) 7.20 (s, 1H).

Preparation of 3-chloro-5-methylbenzaldehyde

To a solution of sodium metal (52 mg, 2.3 mmol) in ethanol was added2-nitropropane (0.23 g, 2.4 mmole) followed by the addition of3-chloro-5-methybenzylbromide (0.5 g, 2.3 mmol). The reaction wasallowed to stir for 3 hours and the precipitate formed was filtered off.The filtrate was concentrated under reduced pressure, redissolved indiethylether and washed with 1N sodium hydroxide (twice), water, anddried over sodium sulfate, filtered and the filtrate was concentratedunder reduced pressure. The resulting residue was purified via silicagel chromatography using 10% dichloromethane and 90% petroleum ether, togive 0.15 g of 3-chloro-5-methylbenzaldehyde (42%). ¹H-NMR (CDCl₃): 2.46(s, 3H) 7.43(s, 1H) 7.56 (s, 1H) 7.68(s, 1H), 9.92 (s, 1H).

3-Chloro-5-fluoro-4-hydroxybenzaldehyde (1.0 gram, 5.7 mmol) in THF (40mL) was heated at reflux for 17 hours with KOH (534 mg, 9.5 mmol, 1.7eq) in water (5 mL) and iodoethane (1 mL, 2.2 eq). The reaction was thentransferred to a separatory funnel with water and extracted withmethylene chloride (thrice, 150 mL each). The combined organic layerswere washed with 10% aqueous HCl (40 mL), dried (MgSO₄), andconcentrated to a viscous orange liquid to yield 1.13 g of3-chloro-4-ethoxy-5-fluorobenzaldehyde (98%). ¹H-NMR (500 MHz, CDCl₃):9.84 (d, J=1.9 Hz, 1H), 7.71 (t, J=1.6 Hz, 1H), 7.53 (dd, J=1.9, 10.7Hz, 1H), 4.37-4.32 (m, 2H), 1.47-1.40 (m, 3H).

4-Ethoxy-3,5-dimethylbenzaldehyde was prepared in a manner similar tothat of 3-chloro-4-ethoxy-5-fluorobenzaldehyde. ¹H-NMR (300 MHz, CDCl₃):9.89 (s, 1H), 7.56 (s, 2H), 3.91 (q, 7 Hz, 1H), 2.34 (s, 6H), 1.44 (t,J=7 Hz, 6H).

4-Isopropoxy-3,5-dimethylbenzaldehyde was prepared in a manner similarto that of 4-Ethoxy-3,5-dimethylbenzaldehyde. ¹H-NMR (300 MHz, CDCl₃):9.88 (s, 1H), 7.55 (s, 2H), 4.31 (q, J=6 Hz, 1H), 2.32 (s, 6H), 1.32 (d,J=6 Hz, 6H).

4-(Cyclopropylmethoxy)-3,5-dimethylbenzaldehyde was prepared in a mannersimilar to that of 4-Ethoxy-3,5-dimethylbenzaldehyde. ¹H-NMR (300 MHz,CDCl₃): 9.87 (s, 1H), 7.55 (s, 2H), 3.69 (d, J=7 Hz, 2H), 2.35 (s, 6H),1.35-1.23 (m, 1H), 0.67-0.060 (m, 2H), 0.35-0.30 (m, 2H).

Preparation of (S)-1-(tert-butoxycarbonyl)-4-oxopyrrolidine-2-carboxylicacid

A solution of(2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid(1.0 eq.) in isopropyl acetate (5 vol) was cooled to 0° C. and TEMPO(0.05 eq.) was added. A solution of bleach (12.5 wt %, 1.2 eq., 2.6 vol)was then slowly added over 1 hour while maintaining the temperature at0-5° C. The mixture was stirred and monitored by HPLC for completion,then aqueous 10% KHSO₄ (2.5 vol) was added, stirred for 10 minutes, andthen the phases were separated. The organic phase was washed withaqueous 5% Na₂SO₃ (2 vol) then brine (1 vol) then dried azeotropicallyand concentrated to afford the title compound as a solid. The solid wastriturated with acetonitrile (1.0 vol) to remove residual color andimpurities. ¹H-NMR (400 MHz, DMSO): δ 4.54 (m, 1H), 3.82 (m, 1H), 3.67(m, 1H); 3.15 (m, 1H); 2.50 (m, 1H, coincides with DMSO); 1.42 and 1.39(2 s rotamers, 9H).

Preparation of(S)-1-(tert-butoxycarbonyl)-4-methylenepyrrolidine-2-carboxylic acid

To a suspension of methyltriphenylphosphonium bromide (2.2 eq.) in2-methyl tetrahydrofuran (3 vol) was added rapidly solid potassiumtert-butoxide (2.3 eq.) maintaining the temperature around 0° C. Thetemperature was kept at +20° C. for 2 hours (a suspension remained) andre-cooled to 0° C. Keeping the temperature below 6° C.,(S)-1-(tert-butoxycarbonyl)-4-oxopyrrolidine-2-carboxylic acid (1 eq.)was added over 40 minutes. The reaction was warmed to room temperatureand stirred for 16 h and then cooled to 0° C. The reaction was quenchedwith saturated NaHCO₃ (5 vol) and water (2 vol) and the aqueous layerwas separated. The organic layer was extracted with saturatedNaHCO₃/water (1.8 vol/1.8 vol) and the combined aqueous layers werefiltered through Celite®. The aqueous layer was acidified with 6 N HCl(2.6 vol) at ambient temperature and extracted twice with isopropylacetate (16 vol, then 8 vol). The organic phase was dried (MgSO₄) andthe solvent removed. The crude product was dissolved in isopropylacetate (10 vol) and extracted with 0.5 M NaOH (10 vol, then 1 vol). Thecombined aqueous layers were acidified at ambient temperature with 6 NHCl to pH=3, and extracted twice with ethyl acetate (10 vol, then 8vol). The combined extracts were dried (Na₂SO₄), the solvent removed andthe crude product was recrystallized from cyclohexane (5 vol) to affordthe title compound. ¹H-NMR (400 MHz, DMSO): δ 12.9, (broad, 1H); 5.00(m, 2H); 4.24 (dt, J=1.9 H, J=7.3 Hz, 1H), 3.91 (m, 2H); 2.98 (m, 1H);2.50 (m, 1H, coincides with DMSO); 1.41 and 1.36 (2 s rotamers, 9H).

Preparation of (5S,8S)-tert-butyl3-(3-chlorophenyl)-1-oxa-2,7-diazaspiro[4.4]non-2-ene-8-carboxylate

A solution of 3-chloro-N-hydroxybenzimidoyl chloride (175 g, 0.919moles) in EtOAc (2.1 L) was added to a solution of (S)-di-tert-butyl4-methylenepyrrolidine-1,2-dicarboxylate (200 g, 0.707 moles) in EtOAc(2.0 L) at room temperature. The mixture was cooled below 10° C. in anice bath, then triethylamine (128 mL, 0.919 moles) was added slowly. Theresultant mixture was stirred overnight then quenched with water (3 L).The phases were separated and the organic phase washed with water (2×1.0L), dried over MgSO₄, and the solvent removed to afford a mixture of thesyn- and anti-spiroisoxazolines as an oil.

The mixture of isomers was dissolved in THF (0.72 L) and cooled to 20°C. Methanesulfonic acid (150 mL) was slowly added maintaining 20 to 30°C. The mixture was stirred at 25° C. and quenched after 7 hours bycarefully adding a solution K₂CO₃ (300 g) in water (1 L). The phaseswere separated and the aqueous phase was extracted with isopropylacetate (1 L). The organic phases were combined and approximately halfof the solvent removed under vacuum. The solution was washed with a 1:1mixture of saturated brine (250 mL) and water (250 mL). The aqueousphase was extracted with isopropyl acetate (200 mL) and the organicphases combined then dried over K₂CO₃ and filtered to afford ahomogeneous solution. The solution volume was made up to 3 L by addingisopropyl acetate and then a solution of oxalic acid (20 g) in isopropylacetate (400 mL) was slowly added. The solid was isolated by filtrationand dried in a vacuum oven. The solid was suspended in isopropyl acetate(1.5 L) and water (1.0 L) then K₂CO₃ was added slowly until the solidsfully dissolved. The organic layer was isolated, dried over K₂CO₃,filtered then a solution of oxalic acid (12.5 g) in isopropyl acetate(250 mL) was added slowly. The solid was isolated by filtration anddried in a vacuum oven to give the spiroisoxazolines as a 98:2anti-:syn-mixture of diastereomers. ¹H-NMR (400 MHz, DMSO-d₆): δ7.67-7.48 (m, 4H), 4.08 (dd, J=7.9, 8.9 Hz, 1H), 3.55 (s, 2H), 3.27 (d,J=4.0 Hz, 2H), 2.46 (dd, J=7.8, 13.8 Hz, 1H), 2.19 (dd, J=9.1, 13.8 Hz,1H), 1.46 (d, J=7.5 Hz, 9H).

Compound X36 (1.0 g, 1.0 eq) was stirred in 20 mL benzene withbenzoylnitromethane (583 mg, 1.0 eq.) and catalytic triethylamine.Phenyl isocyanate (880 uL) was added slowly and stirred for 40 hours.Dark colored precipitate was filtered off and to the filtrate was added2 mL water and the mixture was stirred for 2 hours. Organics wereseparated and concentrated, purified by silica gel chromatography(10-90% ethyl acetate/hexanes gradient) to give 350 mg of Compound X37(25%). (M+H=431.2.) ¹H-NMR (500 MHz, CDCl₃): 8.19 (d, 2H), 7.61 (t, 1H),7.56-7.46 (m, 2H), 4.45-4.36 (m, 1H), 3.99-3.88 (m, 1H), 3.61 (d, 1H),3.39-3.33 (m, 2H), 2.77 (m, 1H), 2.17-2.12 (m, 1H), 1.49 (s, 9H) 1.46(s, 9H).

Compound X37 (1.35 g, 1.0 eq.) was stirred in 20 mL 1/1 TFA/DCM for 2hours. The mixture was concentrated and to it was added 20 mL acetone,20 mL saturated sodium bicarbonate solution, and FMOC-Cl (1.22 g, 1.5eq.). The mixture was stirred for 3 hours and diluted with ethyl acetateand a 2 N HCl solution until aqueous became acidic. The mixture wasstirred, aqueous extracted with ethyl acetate, combined organics, driedover magnesium sulfate, filtered, and concentrated. The concentrate waspurified by silica gel chromatography (100% DCM-10% MeOH/DCM gradient)to give compound X38. (M+H=497.1).

To a solution of 2,3-dihydrobenzofuran 5-carboxaldehyde (1 g, 6.75 mmol)in ethanol (5 mL) was added a 2.4 M of NH₂OH (3.3 mL, 8.1 mmol) solutionand then 1.2 M of Na₂CO₃ (3.3 mL, 4.05 mmol). The resulting solution wasstirred for 2 hours at room temperature (HPLC showed no startingmaterial left). The reaction mixture was diluted with EtOAc, washed withbrine, dried over Na₂SO₄ and concentrated under vacuum. This afforded1.0 g of the product as a white solid. ES-MS 164 as M+1 peak.

To a solution of aldoxime (426 mg, 2.6 mmol) in DMF (5 mL) was added NCS(697 mg, 5.2 mmol). The resulting mixture was stirred for overnight atroom temperature. To the solution was added (S)-di-tert-butyl4-methylenepyrrolidine-1,2-dicarboxylate, compound 1 (600 mg, 2.1 mmol)and then a solution of TEA (0.37 mL, 2.6 mmol) in DMF (2 mL) was addedover 10 minutes. The reaction mixture was stirred for 4 hr at roomtemperature and then heated to 50-60° C. for 2 hours. The reactionmixture was diluted with EtOAc (20 mL) and washed with H₂O, brine, driedover Na₂SO₄, concentrated in vacuo. The crude products were purifiedfrom flash column chromatography eluted with 30% EtOAc/Hexane, to affordS (500-600 mg) (Rf=0.3) and R isomer (150 mg) (Rf=0.2). ES-MS 479 as M+1peak.

B. Synthesis of Exemplary Compounds of Formula I

Certain exemplary compounds of Formula I may be prepared by Method 1 asillustrated below.

Method 1:

Referring to Method 1, the exomethylene compound A1 is deprotected toA2, which is converted to the corresponding Fmoc derivative A3. Reactionof the resin bound aminoalcohol A4 with A3 in the presence of a couplingreagent provides the resin bound product A5. A dipolar addition reactionof A5 with the nitrile oxide 1f, generated in situ, provides the resinbound spiroisoxazoline A6, which is deprotected to provide the resinbound spiroisoxazoline A7. Reaction of A7 with an R₁-carboxylic acid inthe presence of a coupling agent provides A8, wherein R₁ is R₄C(O)—.Cleavage of the spiroisoxazoline from the resin provides the alcohol A9.Oxidation of A9 with an oxidizing reagent such as Dess-Martinperiodinane or sodium hypochlorite in the presence of TEMPO provides thefinal compound A10.

In some instance, R₄ may contain an amine functionality. Where R₄contains a protected amine, deprotection of the protected amine to givea free amine, following by a reaction with an activated acid, provides afurther elaborated R₄. Alternatively, a free amine in R₄ may beconverted to the corresponding p-nitrophenylcarbamate followed byreactions with an amine or alcohol to provide R4 compounds containingcarbamate or urea functionarity.

Preparation of Allyl1-(cyclopropylamino)-2-(6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy)-1-oxohexan-3-ylcarbamate(M1B) Step 1: Allyl1-(cyclopropylamino)-2-hydroxy-1-oxohexan-3-ylcarbamate (M1A)

To a solution of (3S)-3-amino-N-cyclopropyl-2-hydroxyhexanamide (10 g,53.7 mmol), DIEA (28 mL, 161 mmol, 3 eq.) in methylene chloride (250 mL)was added dropewise at 0° C. to a solution of allylchloroformate (6.8mL, 64.4 mmol, 1.2 eq.) in DCM (50 mL). The reaction solution was warmedto room temperature and stirred for 4 hours. Water (300 mL) was thenslowly added followed by aqueous HCl (1.0 N, 300 mL). The phases wereseparated and the organics washed with saturated aqueous NaHCO₃ (300mL), brine (300 mL), dried with MgSO₄, filtered, and concentrated invacuo. The resulting off-white solid was recrystallized from 30% hexanesin EtOAc (120 mL) to yield the title compound M1A as a white solid. Themother liquor was concentrated, in vacuo, and recrystallized from 50%hexanes in EtOAc to yield another 4.04 g of M1A. The mother liquor fromthe second recrystallization was concentrated in vacuo on Celite®, andthe resulting Celite® plug was purified by flash chromatography (IscoCompanion®, SiO₂, DCM to 70% EtOAc in DCM) to give 1.46 g of M1A. Thetotal amount of compound M1A was 13.4 g (yield 93%). (Rf˜0.40 in 1:1DCM:EtOAc, CAM detection).

Step 2: Allyl1-(cyclopropylamino)-2-(6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy)-1-oxohexan-3-ylcarbamatebound resin (M1B)

A 500 mL two neck round bottom flask equipped with an overheadmechanical stirrer and a reflux condenser was charged with M1A (9.08 g,33.6 mmol, 3 eq.), pyridinium p-toluenesulfonate (5.6 g, 22.4 mmol, 2eq.), DHP-resin (10.2 g, 11.2 mmol, Novabiochem, Cat#01-64-0192,loading: 1.1 mmol/g), and dichloroethane (84 mL, [0.4]_(I)). The mixturewas gently stirred at 80° C. for 3 days, before being cooled to 50° C.and filtered. The resin was washed with DCM (200 mL) and the combinedfiltrate were concentrated in vacuo to give the resin M1B, which wasadditionally washed with DCM (twice), DMF (thrice), DCM-MeOH (thrice insuccession), Et₂O, and dried under vacuum overnight to yield a lightbrown resin. The loading of the resin M1B was determined by cleavage ofan aliquot (176 mg) of the resin with 90% aq. TFA. Loading: 0.48 mmol/g.

Preparation of (9H-fluoren-9-yl)methyl2-(1-(cyclopropylamino)-2-hydroxy-1-oxohexan-3-ylcarbamoyl)-4-methylenepyrrolidine-1-carboxylatebound resin (M1E) Step 1: 3-Amino-N-cyclopropyl-2-hydroxyhexanamidebound resin (M1D)

Allyl 1-(cyclopropylamino)-2-hydroxy-1-oxohexan-3-ylcarbamate boundresin M1B (30 g, 1.0 eq.) was swollen with DCM. 1,3-Dimethylbarbituricacid (24.17 g, 12 eq.) and tetrakis(triphenylphosphine)palladium (1.49g, 0.1 eq.) were added and the mixture shaken overnight. The mixture wasfiltered and washed with DMF and DCM to yield the resin M1D.

Step 2: (9H-Fluoren-9-yl)methyl2-(1-(cyclopropylamino)-2-hydroxy-1-oxohexan-3-ylcarbamoyl)-4-methylenepyrrolidine-1-carboxylatebound resin (M1E)

Resin M1D (1.0 g, 1.0 eq.) was stirred in DMF withFMOC-4-exomethyleneproline carboxylic acid (248 mg, 1.1 eq.), HBTU (4.8mL of 0.5 M DMF solution, 5.0 eq.), HOBt (2.4 mL of 1.0 M DMF solution,5.0 eq.), DIEA (836 uL, 10.0 eq.) for 3 hours. The resulting mixture wasdrained and washed with DMF (thrice) and DCM (thrice) to give titlecompound M1E.

Preparation of Fmoc-Protected Isoxazoline Compound Bound Resin (M1F)

The resin M1E (2 g, 0.94 mmol) in THF was shaken with3-chlorobenzaldoxime (5 eq.) and bleach (5% NaOCl) (15 eq.) for 18hours. The resin was then filtered and washed with water, DMF, and DCMto yield the resin compound M1F. An aliquot of the resin was cleaved toprovide a sample for LC-mass analysis (M+1=671).

Preparation of Fmoc Protected Isoxazoline Bound Resin Compound (M1G)

The resin M1F was shaken in 20% piperidine/DMF for 10 minutes, filtered,and washed with DMF and DCM. The THP resin bound spiroisoxazolineproline (0.14 mmol, 0.3 g) was mixed with FMOC-L-3-benzothienyl-ALA(0.56 mmol, 0.25 g), HOBT (0.56 mmol, 0.075 g),N,N-diisopropylethylamine (0.56 mmol, 0.072 g), HBTU (0.56 mmol, 0.21 g)in DMF 2.3 mL and was agitated for 48 hours. The resin was filtered andwashed with DMF, dichloromethane, and ether to yield the resin compoundM1G.

Preparation of7-((S)-3-(benzo[b]thiophen-3-yl)-2-(2-cyclohexylacetamido)propanoyl)-3-(3-chlorophenyl)-N-((3R)-1-(cyclopropylamino)-2-hydroxy-1-oxohexan-3-yl)-1-oxa-2,7-diazaspiro[4.4]non-2-ene-8-carboxamide(M1H)

To the THP-resin bound FMOC protected spiroisoxazoline M1G was added 20%piperidine in DMF (3 mL). The mixture was agitated for 1 hour, filtered,and washed with DMF and dichloromethane. The resin was them mixed withcyclohexylacetic acid (0.56 mmol, 80 mg), HOBT (0.56 mmol, 0.075 g),N,N-diisopropylethylamine (0.56 mmol, 0.072 g), HBTU (0.56 mmol, 0.21 g)in DMF 2.3 mL and was agitated for 48 hr. The resin was filtered andwashed with DMF, dichloromethane, and ether. The resin obtained was thenmixed with a solution of (50:45:5) trifluoroacetic acid,dichloromethane, and triisopropyl silane (3 mL) and was agitatedovernight. The reaction was filtered and washed with dichloromethane.The filtrate was concentrated under vacuum and purified via silica gelchromatography using a gradient of 40% ethyl acetate/60% dichloromethaneto 100% ethyl acetate to produce the alcohol M1H.

EXAMPLE 1 Compound No. 336

To a solution of the hydroxyamide M1H (14 mg, 0.018 mmol) in 0.38 mL ofethyl acetate was added EDC (35 mg, 0.18 mmol) followed by DMSO (0.070mL). The mixture was cooled in an ice bath and dichloroacetic acid (15mg, 0.12 mmol) in ethyl acetate (0.15 mL) was added. The reaction waswarmed to room temperature and allowed to stir for 15 minutes and thencooled in an ice bath and quenched with 1.0 N HCl (0.21 mL). Thesolution was partitioned between ethyl acetate and water. The organicphase was washed with water and dried over sodium sulfate and evaporatedsolvent under vacuum. The resulting residue was purified bychromatography over silica gel using ethyl acetate and hexanes (3:1) aseluant to give Compound No. 336 as a white solid.

Preparation of (9H-fluoren-9-yl)methyl8-((3S)-1-(cyclopropylamino)-2-hydroxy-1-oxohexan-3-ylcarbamoyl)-3-phenyl-1-oxa-2,7-diazaspiro[4.4]non-2-ene-7-carboxylate(M1N)

Step 1: Fmoc Protected Phenyl-Substituted Isoxazoline Bound Resin (M1L)

The resin M1K (2 g, 0.94 mmol) in THF was shaken with the oxime (5 eq.)and bleach (5% NaOCl) (15 eq.) for 18 hours. The resin was then filteredand washed with water, DMF, and DCM to give the Fmoc protectedphenyl-substituted isoxazoline bound resin M1L. An aliquot of resin wascleaved for LC-mass analysis (M+1=637).

The resin M1L (0.47 mmol) was shaken in 20% piperidine/DMF for 10minutes, and then filtered and washed with DMF and DCM. The resultingresin was shaken overnight with a solution of Fmoc-tBG-OH (480 mg 3.0eq.), HOBT (2.82 mL of 0.5 M in DMF, 3.0 eq.), HBTU (2.82 mL of 0.5 M inDMF, 3.0 eq.), and DIEA (0.493 mL, 6.0 eq.). The resin was then filteredand washed with DMF and DCM to give the resin compound M1M, which wasused in next reaction without further purification.

Step 2: Compound M1N

The resin M1M (0.47 mmol) was shaken in 20% piperidine/DMF for 10minutes. The resin was filtered, washed with DMF and DCM. The resultingresin (140 mg, 0.065 mmol) was shaken overnight with benzylisocyanate(176 mg 20.0 eq.), then filtered and washed with DMF and DCM. The resinwas shaken with 90% TFA in water for 30 min. The resulting solution wasconcentrated in vacuo to give the compound M1N (0.065 mmol), (M+1) 661,which was used in next reaction without further purification.

EXAMPLE 2 Compound No. 107

A solution of amide compound M1N in DCM (3 mL) was stirred withDess-Martin Periodinane (54 mg, 2 eq.) and t-BuOH (54 uL) for 1 hour,and then sodium thiosulfate was added to the mixture. The product wasextracted with EtOAc and the combined organic layer was then washed withwater, NaHCO₃, brine and concentrated in vacuo and purified by GilsonPrep HPLC to afford Compound No. 107. (M+1) 659.

EXAMPLE 3 Compound No. 283

Compound 283

The THP resin M1M (0.065 mmol) was shaken in 20% piperidine/DMF for 10minutes, and then filtered and washed with DMF and DCM. The resultingresin was shaken overnight with a solution of 2-(pyridin-3-yl)aceticacid (0.25 mmol 3.0 eq.), HOBT (0.5 mL of 0.5 M in DMF, 3.85 eq.), HBTU(0.5 mL of 0.5 M in DMF, 3.85 eq.), and DIEA (0.5 mmol, 7.69 eq.). Theresin was then filtered and washed with DMF and DCM and was shaken with90% TFA in water for 30 minutes. The resulting solution was concentratedin vacuo to give the hydroxyl amide compound M1P (0.065 mmol) which wasused in the next reaction without further purification. (M+1) 647.

A solution of the hydroxyl amide M1P (0.065 mmol) in DCM (3 mL) wasstirred with Dess-Martin Periodinane (41 mg, 1.5 eq.) and t-BuOH (41uL). After stirred for 1 hour, sodium thiosulfate was added to abovemixture. The product was extracted with EtOAc. The combined organiclayer was then washed with water, NaHCO₃, brine and concentrated invacuo and purified by Gilson Prep HPLC to afford Compound No. 283 (4mg). (M+1) 645.

EXAMPLE 4 Compound No. 61

Compound M1K (750 mg, 1.0 eq.) was stirred in benzene with1-nitropropane (315 uL, 10.0 eq.), and phenylisocyanate (385 uL, 10.0eq.). Triethylamine (5 uL) was added, and the resulting mixture wasshaken overnight, drained, and washed with DMF (thrice) and DCM(thrice). This process was repeated to yield compound M1Q. (M+H=589.0)

Compound M1Q (750 mg, 1.0 eq.) was then shaken in 20% piperidine/DMF for10 minutes. The resin was filtered and washed with DMF (thrice) followedby DCM (thrice). This process was repeated. The resulting resin wasshaken overnight with a solution of(S)-3,3-dimethyl-2-(((S)-tetrahydrofuran-3-yloxy)carbonylamino)butanoicacid (216 mg, 2.5 eq.), HBTU (1.76 mL of 0.5 M in DMF, 3.0 eq.), HOBt(0.88 mL of 1.0 M in DMF, 2.5 eq.), and DIEA (307 uL, 5.0 eq.) in DMF.The resin was then filtered and washed with DMF (thrice) and DCM(thrice) to give compound M1R. (M+H=593.9)

Compound M1R (750 mg, 1.0 eq.) stirred in 1/1 TFA/DCM for 3 hours. Theresin was drained and washed with DCM (thrice). All of the organics wereconcentrated and DCM was added followed by Dess-Martin Periodinane (50mg, 3.0 eq.). The resulting mixture was stirred for 1 hour, 1 N Na₂S₂O₃was added, and stirred again. A racemic mixture of Compound No. 61 waspurified by silica gel chromatography (10-90% ethyl acetate/hexanesgradient) to yield Compound No. 61 as one diastereomer. (M+H=591.8)¹H-NMR (500 MHz, CDCl₃): 7.12 (d, 1H), 6.91 (d, 1H), 5.48 (d, 1H), 5.34(td, 1H), 5.24 (s, 1h), 4.69 (t, 1H), 4.28 (d, 1H), 4.13 (s, 2H),3.93-3.82 (m, 4H), 3.60 (d, 1H), 3.06 (s, 0.5H), 3.03 (s, 0.5H), 2.95(d, 1H), 2.90 (d, 1H), 2.78 (td, 1H), 2.51-2.47 (m, 1H), 2.44-2.34 (m,3H), 2.14-2.10 (m, 1H), 1.94-1.88 (m, 1H), 1.63-1.57 (m, 1H), 1.46-1.36(m, 2H), 1.17 (t, 3H), 0.98 (s, 9H), 0.95-0.83 (m, 5H), 0.59 (dd, 2H)

EXAMPLE 5 Compound No. 146

Compound M1K (50 mg, 1.0 eq.) was stirred in DCM with (Z)-ethyl2-chloro-2-(hydroxyimino)acetate (7.1 mg, 2.0 eq.). To this mixture wasslowly added TEA (6.6 uL, 2.0 eq.) in DCM and the mixture was shaken for3 hours, then drained and washed with DMF (thrice) and DCM (thrice).This process was repeated to give compound M1S (M+H=632.4).

Compound M1S (1.0 g, 1.0 eq.) was shaken in 20% piperidine/DMF for 10minutes. The resin was filtered and washed with DMF (thrice) followed byDCM (thrice). This process was repeated. The resulting resin was shakenovernight with a solution of(S)-3,3-dimethyl-2-(((S)tetrahydrofuran-3-yloxy)carbonylamino)butanoicacid (230 mg 2.0 eq.), HBTU (1.88 mL of 0.5 M in DMF, 2.0 eq.), HOBt(0.94 mL of 1.0 M in DMF, 2.0 eq.), and DIEA (327 uL, 4.0 eq.) in 2 mLDMF. The resin was then filtered and washed with DMF (thrice) and DCM(thrice) to give compound M1T (M+H=638.0).

Compound M1T (750 mg, 1.0 eq.) was shaken in THF with KOTMS (133 mg, 3.0eq.) for 3 hours. The mixture was then drained and washed with THF/water(1/1), THF, DMF, and DCM (thrice each) to give compound M1U.(M+H=609.5).

Compound M1U (250 mg, 1.0 eq.) was shaken overnight with a solution ofethylamine (22 mg 3.0 eq.), HBTU (0.54 mL of 0.5 M in DMF, 3.0 eq.),HOBt (0.27 mL of 1.0 M in DMF, 3.0 eq.), and DIEA (47 uL, 3.0 eq.) inDMF. The resin was then filtered and washed with DMF (thrice) and DCM(thrice) to give compound M1V. (M+H=637.2).

Compound M1V (0.4 g, 1.0 eq.) was stirred in 1/1 TFA/DCM for 2 hours andthen drained and washed with DCM (thrice). The organic phases werecombined and dried, and to it was added DCM followed by Dess-MartinPeriodinane (97 mg, 3.0 eq.). The solution was stirred for 1 hour and toit was added 1 N Na₂S₂O₃ and the mixture was further stirred. Thesolution was purified by silica gel chromatography (10-90% ethylacetate/hexanes gradient) to yield 6.1 mg of Compound No. 146.(M+H=635.0) ¹H-NMR (CDCl₃): 5.5-5.2 (m, 2H), 5.1-5.0 (m, 1H), 4.9-4.7(m, 2H), 4.5-4.2 (m, 3H), 4.1 (m, 1H), 3.9-3.7 (m, 3H), 3.6-3.5 (m, 2H),3.5-3.2 (m, 2H), 2.8-2.4 (m, 2H), 2.1 (m, 1H), 2.0-1.8 (m, 3H), 1.8-1.5(m, 3H), 1.5-1.3 (m, 3H), 1.3-1.2 (m, 2H), 1.0 (s, 9H), 0.9 (t, 3H), 0.8(m, 2H), 0.6 (m, 2H).

The following compounds of Formula I were also produced according toMethod 1 and the preparations described thereunder.

TABLE 1 Additional Compounds of Formula I Produced by Method 1. Com-pound Starting Starting Starting No. Material for P¹ Material for C¹Material for R₃ 7 N—FMOC-L-tert- 3-(4-fluoro- 3-chlorobenzene-butylglycine phenyl)propanoic carbaldehyde acid oxime 12 N—FMOC-L-tert-Acetic acid 3-chlorobenzene- butylglycine carbaldehyde oxime 14N—FMOC-L-tert- cyclopentylmethanol 3-chlorobenzene- butylglycinecarbaldehyde oxime 24 N-((S)- N/A 3-Fluorobenzene- tetrahydrofuran-3-carbaldehyde yloxy)carbonyl)-L- oxime tert-butylglycine 27N—FMOC-L-tert- cyclobutylmethanol 3-chlorobenzene- butylglycinecarbaldehyde oxime 29 N—FMOC-L-tert- 2-(1-methylcyclo- 3-chlorobenzene-butylglycine hexyl)acetic acid carbaldehyde oxime 30 N—FMOC-L-tert-(S)-5-oxo-1-(thio- 3-chlorobenzene- butylglycine phen-2-ylmeth-carbaldehyde yl)pyrrolidine-2- oxime carboxylic acid 33 N—FMOC-L-tert-cyclohexyl 3-chlorobenzene- butylglycine isocyanate carbaldehyde oxime34 N—FMOC-L-tert- 5-hydroxypentan-2- 3-chlorobenzene- butylglycine onecarbaldehyde oxime 37 N—FMOC-L-tert- 2-(tetrahydro-2H- 3-chlorobenzene-butylglycine pyran-4-yl)acetic carbaldehyde acid oxime 39 N—FMOC-L-tert-2-chlorobenzyl 3-chlorobenzene- butylglycine chloroformate carbaldehydeoxime 44 N—FMOC-L-tert- 4-oxo-pentanoic 3-chlorobenzene- butylglycineacid carbaldehyde oxime 53 N/A 2-(1-(2,6-dichloro- Benzaldoximebenzyl)piperidin-4- yl)acetic acid 61 N-((S)- N/A Nitropropanetetrahydrofuran-3- yloxy)carbonyl)-L- tert-butylglycine 71N—FMOC-L-tert- (R)-2,3-dihydro- 3-chlorobenzene- butylglycinebenzo[b][1,4]di- carbaldehyde oxine-2-carboxylic oxime acid 72N—FMOC-L-tert- 2-cyclopentylacetic 3-chlorobenzene- butylglycine acidcarbaldehyde oxime 75 N—FMOC-L-tert- 2-(2,4- 3-chlorobenzene-butylglycine dimethylthiazol-5- carbaldehyde yl)acetic acid oxime 76N—FMOC-L-tert- Cyclopropyl 3-chlorobenzene- butylglycine isocyanatecarbaldehyde oxime 85 N—FMOC-L-tert- 2-Fluoroethyl 3-chlorobenzene-butylglycine chloroformate carbaldehyde oxime 92 N—FMOC-L-tert-2-(tetrahydro-2H- 3-chlorobenzene- butylglycine pyran-4-yl)aceticcarbaldehyde acid oxime 93 N—FMOC-L-tert- cyclohexane- 3-chlorobenzene-butylglycine carboxylic acid carbaldehyde oxime 94 N—FMOC-L-tert-2-aminoacetamide 3-chlorobenzene- butylglycine carbaldehyde oxime 102N—FMOC-L-tert- 2-(3-fluoro-4- 3-chlorobenzene- butylglycinemethylphenyl)acetic carbaldehyde acid oxime 107 N—FMOC-L-tert- Benzylisocyanate Benzaldoxime butylglycine 108 N—FMOC-L-tert-cis-4-methoxycyclo- 3-chlorobenzene- butylglycine hexanecarboxyliccarbaldehyde acid oxime 110 N—FMOC-L-tert- Benzyl 3-Chloro-4,6-dimeth-butylglycine chloroformate oxybenzaldoxime 112 N—FMOC-L-tert-2-(tetrahydro-2H- 2-nitro-1-phenyl butylglycine pyran-4-yl)aceticethanone acid 118 N—FMOC-L-tert- 2-(4-fluoro- 3-Chlorobenzene-butylglycine phenyl)ethanol carbaldehyde oxime 119 N—FMOC-L-tert-tert-Butyl isocyanate 2-nitro-1-phenyl butylglycine ethanone 122N—FMOC-L-tert- 3-Fluorobenzyl 3-Chlorobenzene- butylglycine isocyanatecarbaldehyde oxime 123 N—FMOC-L-tert- Ethyl isocyanate 3-Chlorobenzene-butylglycine carbaldehyde oxime 124 N—FMOC-O- 2-cyclohexylacetic3-Chlorobenzene- Methyl-L- acid carbaldehyde oxime Threonine 125(2R,3S)-N— 2-cyclohexylacetic 3-chlorobenzene- FMOC-2-Amino-3- acidcarbaldehyde phenyl-butyric acid oxime 128 N—FMOC-L-tert-4-(1H-pyrrole-2,5- 3-chlorobenzene- butylglycine dione)phenylcarbaldehyde isocyanate oxime 135 N—FMOC-L-tert- 1-isopropyl-4-oxo-3-chlorobenzene- butylglycine 1,4-dihydroquino- carbaldehydeline-3-carboxylic oxime acid 139 N—FMOC-L-tert- (R)-2-hydroxy-2-3-chlorobenzene- butylglycine phenylpropanoic carbaldehyde acid oxime146 N-((S)- N/A 2-Chloro-2- tetrahydrofuran-3- hydroximinoaceticyloxy)carbonyl)-L- aicd ethyl ester tert-butylglycine (chlorooxime) 152N—FMOC-L-tert- (tetrahydrofuran-3- 3-chlorobenzene- butylglycineyl)methanol carbaldehyde oxime 154 N-Alloc-L-tert- N/A 4-Fluorobenzene-butylglycine carbaldehyde oxime 155 N—FMOC-L-tert- 2-(5-fluoro-2-3-chlorobenzene- butylglycine methylphenyl)acetic carbaldehyde acidoxime 156 N—FMOC-L-tert- Isobutylamine 3-chlorobenzene- butylglycinecarbaldehyde oxime 159 N—FMOC-L-tert- 2-(thiophen-3- 3-chlorobenzene-butylglycine yl)ethanol carbaldehyde oxime 160 N—CBZ-L-tert- N/A4-Fluorobenzene- butylglycine carbaldehyde oxime 161 N—FMOC-L-tert-5-acetamido-2- 3-chlorobenzene- butylglycine acetylthiophene-3-carbaldehyde carboxylic acid oxime 164 N—CBZ-L-tert- N/A2-Chlorobenzene- butylglycine carbaldehyde oxime 167 N—FMOC-L-tert-(2-methylpyridin-3- 3-chlorobenzene- butylglycine yl)methanolcarbaldehyde oxime 173 N—FMOC-L-tert- 2,2- 3-chlorobenzene- butylglycinedifluoroethylamine carbaldehyde oxime 174 N—FMOC-L-tert- m-tolylmethanol3-chlorobenzene- butylglycine carbaldehyde oxime 180 N—FMOC-L-tert-Acetic acid Nitroethane butylglycine 183 N—CBZ-L-tert- N/A3-Fluorobenzene- butylglycine carbaldehyde oxime 185 N—FMOC-L-3-2-cyclohexylacetic 3-chlorobenzene- Thienyl-Alanine acid carbaldehydeoxime 193 N—FMOC-L-tert- Isopropyl 3-chlorobenzene- butylglycinechloroformate carbaldehyde oxime 199 N—CBZ-L-tert- N/A 9-Anthraldehydebutylglycine oxime 201 N—FMOC-L-tert- (3-methoxy- 3-chlorobenzene-butylglycine phenyl)methanol carbaldehyde oxime 203 N—FMOC-L-tert-(3,5-difluoro- 3-chlorobenzene- butylglycine phenyl)methanolcarbaldehyde oxime 205 N—FMOC-L-tert- Benzyl isocyanate 3-chlorobenzene-butylglycine carbaldehyde oxime 207 N—FMOC-L- Ethyl chloroformate3-chlorobenzene- Glycine carbaldehyde oxime 208 N—CBZ-L-tert- N/A2-Naphthaldehyde butylglycine oxime 209 N—FMOC-L-tert- (3-fluoro-3-chlorobenzene- butylglycine phenyl)methanol carbaldehyde oxime 210N—FMOC-L-tert- 2-chlorobenzyl 3-Chloro-4,6- butylglycine chloroformatedimethoxy- benzaldoxime 213 N—FMOC-4- 2-cyclohexylacetic3-chlorobenzene- Methoxy-L- acid carbaldehyde Phenylalanine oxime 216N—FMOC-L-tert- 5-oxo-1-(thiophen- 3-chlorobenzene- butylglycine2-ylmeth- carbaldehyde yl)pyrrolidine-3- oxime carboxylic acid 235N—CBZ-L-tert- N/A nitrobutane butylglycine 237 N—FMOC-L-tert-3-(2-methyl-1H- 3-chlorobenzene- butylglycine imidazol-1- carbaldehydeyl)propanoic acid oxime 241 N—FMOC-L-tert- (S)-1-isopropyl-5-3-chlorobenzene- butylglycine oxopyrrolidine-2- carbaldehydecarboxylicacid oxime 242 N—FMOC-L-tert- N—FMOC-L- Piperonal oximebutylglycine cyclohexylglycine followed by 2- pyrazine carboxylic acid243 N-((S)- N/A nitrobutane tetrahydrofuran-3- yloxy)carbonyl)-L-tert-butylglycine 249 N—FMOC-L-tert- cyclohexanemethyl Benzaldoximebutylglycine isocyanate 254 N—FMOC-L-tert- 1-(thiophen-2-3-chlorobenzene- butylglycine yl)propan-2-ol carbaldehyde oxime 259N—FMOC-L-tert- 3,4,5-trimethoxy- 3-chlorobenzene- butylglycine benzylisocyanate carbaldehyde oxime 260 N—FMOC-L-tert- 2-methoxyethyl3-chlorobenzene- butylglycine chloroformate carbaldehyde oxime 261N—FMOC-L-tert- benzyl 4-isocyanato- 3-chlorobenzene- butylglycinepiperidine-1- carbaldehyde carboxylate oxime 262 N/A 4-nitrophenyl3-chlorobenzene- choroformate carbaldehyde oxime 276 2-((3S,4aS,8aS)-3-N/A 3-chlorobenzene- (tert-butylcarba- carbaldehyde moyl)octa-hydro-oxime isoquinolin-2(1H)- yl)acetic acid 278 N—FMOC-L-tert- benzyl2-(4-Methoxy- butylglycine chloroformate phenoxy)benzenecarb aldehydeoxime 283 N—FMOC-L-tert- 2-(pyridin-3- Benzaldoxime butylglycineyl)acetic acid 287 N—FMOC-L-tert- 2-(3-methoxy- 3-chlorobenzene-butylglycine phenyl)acetic acid carbaldehyde oxime 288 N—FMOC-L-tert-1-Naphthyl Benzaldoxime butylglycine isocyanate 289 N—FMOC-2-2-cyclohexylacetic 3-chlorobenzene- Trifluoromethyl-L- acid carbaldehydePhenylalanine oxime 291 N—FMOC-L-tert- spiro[indene-1,4′-3-chlorobenzene- butylglycine piperidin]-3(2H)- carbaldehyde one oxime294 N—FMOC-L-tert- 2-cyclohexylacetic 3-chlorobenzene- butylglycine acidcarbaldehyde oxime 308 N—FMOC-L-tert- (tetrahydro-2H- 3-chlorobenzene-butylglycine pyran-2-yl)methanol carbaldehyde oxime 311 N—FMOC-L-tert-2-(pyrrolidine-1- 3-chlorobenzene- butylglycine carbonyl)cyclo-carbaldehyde hexanecarboxylic oxime acid 313 N/A benzyl isocyanate3-Chloro-4,6- dimethoxybenzald oxime 317 N—FMOC-L-tert-2-(1-oxoisoindolin- 3-chlorobenzene- butylglycine 2-yl)propanoic acidcarbaldehyde oxime 324 N—FMOC-L-tert- (R)-3-(1- 3-chlorobenzene-butylglycine cyanoethyl)benzoic carbaldehyde acid oxime 329N—FMOC-L-tert- cyclohexylacetic Nitropropane butylglycine acid 331N—FMOC-L-tert- acetic acid 4-Fluorobenzene- butylglycine carbaldehydeoxime 333 N—FMOC-L-tert- (S)-1- 3-Chlorobenzene- butylglycinemethylbenzylamine carbaldehyde oxime 334 N—FMOC-L-tert- (S)-2-methyl-3-3-chlorobenzene- butylglycine phenylpropanoic carbaldehyde acid oxime336 N—FMOC-L-3- 2-cyclohexylacetic 3-chlorobenzene- Benzothienyl- acidcarbaldehyde Alanine oxime 338 N—FMOC-2- 2-cyclohexylacetic3-chlorobenzene- Fluoro-L- acid carbaldehyde Phenylalanine oxime 340N—CBZ-L-tert- N/A 4- butylglycine Phenylbenzene- carbaldehyde oxime 341N-((S)- N/A 2-chloro-2- tetrahydrofuran-3- hydroximinoaceticyloxy)carbonyl)-L- acid ethyl ester tert-butylglycine (chlorooxime)followed by ester hydrolysis and coupling of ethylamine 342 N/A pyridine3-methanol 3-chlorobenzene- carbaldehyde oxime 345 N-(5-methyl-3- N/A3-chlorobenzene- nitroprydinyl)-L- carbaldehyde tert-butylglycine oxime349 N—FMOC-L-tert- 2-(4-fluoro- 3-chlorobenzene- butylglycinephenyl)ethanol carbaldehyde oxime 352 N-((S)- N/A Pyridine-4-tetrahydrofuran-3- aldoxime yloxy)carbonyl)-L- tert-butylglycine 357N—FMOC-L-tert- pyridin-4- 3-chlorobenzene- butylglycine ylmethanolcarbaldehyde oxime 358 N-((S)- N/A 4-Trifluoromethoxy-tetrahydrofuran-3- benzenecarbaldehyde yloxy)carbonyl)-L- oximetert-butylglycine 365 N—CBZ-L-tert- N/A 4-Trifluoromethoxy- butylglycinebenzenecarbaldehyde oxime 367 N/A 3,4,5-trimethoxy- Benzaldoxime benzylisocyanate 373 N—FMOC-L-tert- 3-(pyridin-2- 3-chlorobenzene-butylglycine yl)propan-1-ol carbaldehyde oxime 374 N—FMOC-L-tert-tetrahydro-2H- 3-chlorobenzene- butylglycine pyran-4-ol carbaldehydeoxime 377 N—FMOC-L-tert- (S)-1-(3-chloro- 3-chlorobenzene- butylglycinebenzyl)-5-oxo- carbaldehyde pyrrolidine-2- oxime carboxylic acid 378N—FMOC-L-tert- pyridin-2- 3-chlorobenzene- butylglycine ylmethanolcarbaldehyde oxime 379 N—FMOC-L-tert- isopropyl isocyanate3-chlorobenzene- butylglycine carbaldehyde oxime 381 N—FMOC-L-tert-4-oxo-3,4-dihydro- 3-chlorobenzene- butylglycine phthalazine-1-carbaldehyde carboxylic acid oxime 383 N—CBZ-L-tert- N/A Nitropropanebutylglycine 387 N—FMOC-L-tert- (3R,3aS,6aR)- 3-chlorobenzene-butylglycine hexahydrofuro[2,3- carbaldehyde b]furan-3-ol oxime 389N—FMOC-L-tert- 3-(pyridin-3- 3-chlorobenzene- butylglycineyl)propan-1-ol carbaldehyde oxime 390 N—FMOC-L-tert- cyclohexylacetic2-nitro-1-phenyl butylglycine acid ethanone 398 N-((S)- N/A4-Fluorobenzene- tetrahydrofuran-3- carbaldehyde yloxy)carbonyl)-L-oxime tert-butylglycine 400 N—FMOC-L-tert- N—FMOC-L- nitrobutanebutylglycine cyclohexylglycine followed by 2- pyrazine carboxylic acid402 N—FMOC-L-tert- 2-(5-oxo-2-(thio- 3-chlorobenzene- butylglycinephen-2-yl)cyclo- carbaldehyde pent-1-enyl)acetic oxime acid 407N—FMOC-L-tert- ethyl chloroformate 3-chlorobenzene- butylglycinecarbaldehyde oxime 417 N—FMOC-L-tert- N—FMOC-L-tert- 4-Fluorobenzene-butylglycine butylglycine carbaldehyde followed by 2- oxime pyrazinecarboxylic acid 427 N—FMOC-L- ethyl chloroformate 3-chlorobenzene-Phenylalanine carbaldehyde oxime 431 N—FMOC-L-tert- 2-o-tolylacetic acid3-chlorobenzene- butylglycine carbaldehyde oxime 432 N—FMOC-L-tert-N-methyl 3-chlorobenzene- butylglycine ethylamine carbaldehyde oxime 437N—FMOC-S-tert- 2-cyclohexylacetic 3-chlorobenzene- Butyl-L-Cysteine acidcarbaldehyde oxime 450 N—FMOC-L-tert- N—FMOC-L-tert- Piperonal oximebutylglycine butylglycine followed by 2- pyrazine carboxylic acid 454N—FMOC-L-tert- 2-(quinolin-8- 3-chlorobenzene- butylglycineylthio)acetic acid carbaldehyde oxime 459 N—FMOC-L- 2-cyclohexylacetic3-chlorobenzene- Norleucine acid carbaldehyde oxime 462 N—FMOC-L-tert-cyclohexylacetic Piperonal oxime butylglycine acid 463 N—FMOC-L-tert-2-phenylethanol 3-chlorobenzene- butylglycine carbaldehyde oxime 465N-(Cyclo- N/A 4-Fluorobenzene- pentylformoyl)- carbaldehyde L-tert-oxime butylglycine 467 N—FMOC-L-tert- 2-(bi- 3-chlorobenzene-butylglycine cyclo[2.2.1]heptan- carbaldehyde 2-yl)acetic acid oxime 471N—FMOC-L-tert- p-tolylmethanol 3-chlorobenzene- butylglycinecarbaldehyde oxime 474 N—FMOC-L-tert- 2-methyl-3-(3- 3-chlorobenzene-butylglycine methyl-1H-pyrazol- carbaldehyde 1-yl)propanoic acid oxime477 N—FMOC-L-tert- (S)-1-methoxy-3,3- 3-chlorobenzene- butylglycinedimethylbutan-2- carbaldehyde amine oxime 484 N—FMOC-L-tert- SuccinicAnhydride 3-chlorobenzene- butylglycine carbaldehyde oxime 487N—FMOC-L-tert- 2-(6-methoxy-3- 3-chlorobenzene- butylglycineoxo-2,3-dihydro-1H- carbaldehyde inden-1-yl)acetic oxime acid 487N—FMOC-L-tert- 2-(3-oxo-2,3- 3-chlorobenzene- butylglycinedihydro-1H-inden-1- carbaldehyde yl)acetic acid oxime 492 N—FMOC-L-tert-tert-Butyl isocyanate 3-chlorobenzene- butylglycine carbaldehyde oxime497 N—FMOC-L-tert- pyridin-3- 3-chlorobenzene- butylglycineylmethylamine carbaldehyde oxime 503 N—FMOC-L-tert- trans-4-methoxy-3-chlorobenzene- butylglycine cyclohexane- carbaldehyde carboxylic acidoxime 504 N—FMOC-L-tert- 3-(pyridin-3- 3-chlorobenzene- butylglycineyl)propanoic acid carbaldehyde oxime 505 N—FMOC-L-tert- 3-(2,5-dioxo-3-chlorobenzene- butylglycine imidazolidin-4- carbaldehyde yl)propanoicacid oxime 512 N—FMOC-L-tert- (2-fluoro- 3-chlorobenzene- butylglycinephenyl)methanol carbaldehyde oxime 515 N—FMOC-L-tert- tetrahydro-2H-3-chlorobenzene- butylglycine pyran-3-ol carbaldehyde oxime 517N—FMOC-L-tert- N/A Nitroethane butylglycine 518 N—FMOC-L-tert-(S)-1-(3-methyl- 3-chlorobenzene- butylglycine benzyl)-5-oxo-carbaldehyde pyrrolidine-2- oxime carboxylic acid 520 N—FMOC-L-tert-benzyl Benzaldoxime butylglycine chloroformate 523 N—FMOC-L-tert-tetrahydro-2H- 3-chlorobenzene- butylglycine pyran-4-carboxyliccarbaldehyde acid oxime 526 N—FMOC-L-tert- benzyl 3-chlorobenzene-butylglycine chloroformate carbaldehyde oxime 528 N—FMOC-L-tert-3-(1H-indazol-1- 3-chlorobenzene- butylglycine yl)propanoic acidcarbaldehyde oxime 532 N—FMOC-L-tert- 3-methylbutanoic 3-chlorobenzene-butylglycine acid carbaldehyde oxime 533 N/A N/A 4-Fluorobenzene-carbaldehyde oxime 538 N—FMOC-L-tert- 2-cyano-2-methyl-3-3-chlorobenzene- butylglycine phenylpropanoic carbaldehyde acid oxime544 N—FMOC-L-tert- 3-(1H-benzo[d]im- 3-chlorobenzene- butylglycineidazol-1-yl)-2- carbaldehyde methylpropanoic oxime acid 547N—FMOC-L-tert- N—FMOC-L- Nitropropane butylglycine cyclohexylglycinefollowed by 2- pyrazine carboxylic acid 553 N—FMOC-L-tert- 2-(2,6-dioxo-3-chlorobenzene- butylglycine 1,2,3,6-tetrahydro- carbaldehydepyrimidin-4- oxime yl)acetic acid 557 N—FMOC-L-tert- (1R,6S)-6-(meth-3-chlorobenzene- butylglycine oxycarbonyl)cyclo- carbaldehydehex-3-enecarboxylic oxime acid 558 N—FMOC-L-tert- phenyl isocyanate3-chlorobenzene- butylglycine carbaldehyde oxime 559 N—FMOC-L-tert-tert-Butyl isocyanate Nitropropane butylglycine 561 N—FMOC-L-tert-(2,5-difluoro- 3-chlorobenzene- butylglycine phenyl)methanolcarbaldehyde oxime 563 N—FMOC-L-tert- Pyridine 3-methanol3-chlorobenzene- butylglycine carbaldehyde oxime 566 N—FMOC-L-tert-2-(tetrahydro-2H- Nitropropane butylglycine pyran-4-yl)acetic acid 576N—FMOC-L-tert- 3-pyridyl isocyanate 3-chlorobenzene- butylglycinecarbaldehyde oxime 580 N—FMOC-L-tert- ethyl isocyanate Benzaldoximebutylglycine 582 N—FMOC-L-tert- 2-(thiophen-2- 3-chlorobenzene-butylglycine yl)ethanol carbaldehyde oxime 583 N—FMOC-L-tert- benzylisocyanate 3-chlorobenzene- butylglycine carbaldehyde oxime

All starting materials for R₃ listed in Table 1 and all other tablesherein were either commercially available (nitro or oxime) or readilyprepared from corresponding aldehyde precursors.

Additionally, Compound Nos. 20, 22, 53, 81, 103, 116, 166, 187, 189,194, 197, 200, 220, 223, 226, 245, 252, 271, 204, 307, 319, 339, 354,360, 361, 371, 392, 393, 435, 449, 506, 514, 531, and 585 were alsoproduced by using Method 1.

Certain other compounds of the invention may be prepared as illustratedby Method 2.

Method 2:

Referring to Method 2, the protected spiroisoxazoline B1 is deprotectedto B2 which in turn is converted to the Fmoc derivative B3. Reaction ofB3 with the resin bound aminoalcohol A4 provides the resin boundspiroisoxazoline A6 which is converted to A10 as described in Method 1.

EXAMPLE 6 Compound No. 281

Compound M2A (5.0 g, 1.0 eq.) was stirred in 100 mL acetonitrile and tothis mixture was added ditertbutyldicarbonate (9.6 g, 2.0 eq.),dimethylaminopyridine (537 mg, 0.2 eq.), and triethylamine (6.13 mL, 2.0eq.) and stirred overnight. The resulting mixture was concentrated,ethyl acetate was added, and the mixture was washed with 1.0 N HCl,dried over sodium sulfate, concentrated, and purified by silica gelchromatography (10-30% ethyl acetate/hexanes gradient) to yield compoundM2B. (M+H=284.0) ¹H-NMR (CDCl₃): 5.0 (m, 2H), 4.3-4.5 (m, 1H), 4.0-4.1(m, 2H), 2.9-3.0 (m, 1H), 2.5-2.6 (d, 1H), 1.5 (s, 3/9 of 18H), 1.4 (s,6/9 of 18H).

Compound M2B (2.0 g, 1.0 eq.) stirred in 35 mL DCM with benzaldoxime(2.67 g, 2.0 eq.). The solution was cooled on an ice bath and to thisbleach (5% NaOCl) (34.9 mL) was slowly added. The mixture was thenwarmed to room temperature and stirred for 2 hours. The aqueous layerwas separated and extracted with DCM twice. The organics were combinedand dried over magnesium sulfate, filtered and concentrated. Purifiedvia silica gel chromatography (5-30% ethyl acetate/hexanes gradient)yielded compound M2C. (M+H=403.1) ¹H-NMR (500 MHz, CDCl3): 7.64-7.63 (m,2H), 7.41-7.40 (m, 3H), 4.43-4.37 (t, 1H), 3.94-3.85 (dd, 1H), 3.62 (t,1H), 3.44-3.38 (m, 1H), 3.29-3.24 (m, 1H), 2.74 (m, 1H), 2.14-2.10 (m,1H), 1.49 (s, 9H), 1.46 (s, 9H).

Compound M2C was stirred in 1/1 TFA/DCM for 3 hours. The mixture wasconcentrated. To the concentrated mixture was added 17 mL DMF, 5 mLwater, sodium carbonate (713 mg, 2.5 eq.), FMOC-OSu (951 mg, 1.05 eq.)and stirred 3 hours. Then, ethyl acetate was added and the resultingmixture was washed with 1.0 N HCl followed by brine. It was dried overmagnesium sulfate, filtered and concentrated to yield compound M2D.(M+H=468.9).

Compound M2D (1.26 g, 2.0 eq.) was stirred in DMF with M1D (2.5 g, 1.0eq.), HBTU (12 mL of 0.5 M in DMF, 5.0 eq.), HOBt (6 mL of 1.0 M in DMF,5.0 eq.), and Hünig's base (2.09 mL. 10.0 eq.) overnight. The mixturewas drained and washed with DMF (thrice) and DCM (thrice) to yieldcompound M1L. (M+H=637.0).

Compound M1L (0.4 g, 1.0 eq.) was shaken in 20% piperidine/DMF for 10minutes before being filtered and washed with DMF (thrice) followed byDCM (thrice). This process was repeated. The resulting resin was shakenovernight with a solution of FMOC-tert-butylglycine (200 mg 3.0 eq.),HBTU (1.15 mL of 0.5 M in DMF, 3.0 eq.), HOBt (0.58 mL of 1.0 M in DMF,3.0 eq.), and DIEA (167 uL, 5.0 eq.) in 2 mL DMF. The resin was thenfiltered and washed with DMF (thrice) and DCM (thrice) to give compoundM1M. (M+H=750.1).

Compound M1M (0.4 g, 1.0 eq.) was shaken in 20% piperidine/DMF for 10minutes and the resin was filtered and washed with DMF (thrice) followedby DCM (thrice). This process was repeated to give Compound M2H.

Compound M2H (0.4 g, 1.0 eq.) was shaken overnight with a solution ofFMOC-cyclohexylglycine (218 mg 3.0 eq.), HBTU (1.15 mL of 0.5 M in DMF,3.0 eq.), HOBt (0.58 mL of 1.0 M in DMF, 3.0 eq.), and DIEA (167 uL, 5.0eq.) in 2 mL DMF. The resin was then filtered and washed with DMF(thrice) and DCM (thrice). The resin was then treated with 20%piperidine/DMF for 10 minutes. The resin was filtered and washed withDMF (thrice) followed by DCM (thrice). This process was repeated to giveCompound M2I.

Compound M2I (0.4 g, 1.0 eq.) was shaken overnight with a solution ofpyrazine carboxylic acid (71 mg, 3.0 eq.), HBTU (1.15 mL of 0.5 M inDMF, 3.0 eq.), HOBt (0.58 mL of 1.0 M in DMF, 3.0 eq.), and DIEA (167uL, 5.0 eq.) in 2 mL DMF. The resin was then filtered and washed withDMF (thrice) and DCM (thrice) to give compound M2J. (M+H=772.9).

Compound M2J (0.4 g, 1.0 eq.) was stirred in 1/1 TFA/DCM for 2 hours.The resin was drained and washed with DCM (thrice). The result wasconcentrated all organics and added DCM followed by Dess MartinPeriodinane (97 mg, 3.0 eq.). Stirred for 1 hour and added 1N Na₂S₂O₃and stirred. The solution was purified by silica gel chromatography(10-90% ethyl acetate/hexanes gradient) to yield 42 mg of Compound No.281. (M+H=771.0). ¹H-NMR (500 MHz, CDCl₃): 9.38 (d, 1H), 8.75 (d, 1H),8.56 (t, 1H), 8.31 (d, 1H), 7.64-7.62 (m, 2H), 7.42-7.38 (m, 3H), 7.33(d, 1H), 7.15 (s, 1H), 6.89 (d, 1H), 5.45-5.41 (m, 1H), 4.85 (t, 1H),4.69 (d, 1H), 4.57-4.54 (m, 1H), 4.26 (d, 1H), 3.76 (d, 1H), 3.46-3.35(m, 2H), 2.82 (td, 1H), 2.56 (d, 2H), 1.96-1.87 (m, 2H), 1.76 (m, 4H),1.65-1.59 (m, 2H), 1.48-1.42 (m, 2H), 1.24 (m, 2H), 1.09 (m, 2H), 0.97(s, 9H), 0.93 (t, 2H), 0.88-0.84 (m, 2H), 0.65 (t, 2H).

Listed below in Table 2 are additional compounds of Formula I preparedby Method 2.

TABLE 2 Additional Compounds of Formula I Produced by Method 2. Com-pound Starting Starting Starting No. Material for P¹ Material for C¹Material for R₃ 40 N-((S)- N/A 2,6-Dichloro- tetrahydrofuran-3-benzaldoxime yloxy)carbonyl)-L- tert-butylglycine 51 N/A 1H-pyrrole-2-Piperonal carboxylic acid oxime 80 N/A 1H-pyrrole-2- Benzaldoximecarboxylic acid 101 N—FMOC-L-tert- 1-Naphthylsulfonyl Benzaldoximebutylglycine chloride 147 N—FMOC-L-tert- N—FMOC-L- 2,6-Dichloro-butylglycine cyclohexylglycine benzaldoxime followed by 2-pyrazinecarboxylic acid 151 N-Alloc-L-tert- N/A Benzaldoxime butylglycine 202N-((S)- N/A Benzaldoxime tetrahydrofuran-3- yloxy)carbonyl)-L-tert-butylglycine 228 N—FMOC-L-tert- Acetic acid Benzaldoximebutylglycine 281 N—FMOC-L-tert- N—FMOC-L- Benzaldoxime butylglycinecyclohexylglycine followed by 2-pyrazine carboxylic acid 325N—FMOC-L-tert- Acetic acid Piperonal butylglycine oxime 327N-Alloc-L-tert- N/A Piperonal butylglycine oxime 343 N-((S)- N/APiperonal tetrahydrofuran-3- oxime yloxy)carbonyl)-L- tert-butylglycine428 N—FMOC-L-tert- N—FMOC-L- Benzaldoxime butylglycine cyclohexylglycinefollowed by 2-pyrazine carboxylic acid 464 N/A 1H-pyrrole-2- Piperonalcarboxylic acid oxime 491 N-((S)- N/A Benzaldoxime tetrahydrofuran-3-yloxy)carbonyl)-L- tert-butylglycine 527 N-((S)- N/A Piperonaltetrahydrofuran-3- oxime yloxy)carbonyl)-L- tert-butylglycine 536N—FMOC-L-tert- 1-Naphthylsulfonyl Piperonal butylglycine chloride oxime570 N—FMOC-L-tert- Acetic acid Piperonal butylglycine oxime 578N—FMOC-L-tert- Acetic acid Benzaldoxime butylglycine 584 N/A1H-pyrrole-2- Benzaldoxime carboxylic acid

Certain other compounds of Formula I may be prepared as illustrated byMethod 3.

Method 3:

Referring to Method 3, the resin bound Fmoc exomethylene compound A5,prepared as in Method 1, is deprotected to give C1. Reaction of C1 withan R₁ carboxylic acid in the presence of a coupling reagent provides C2wherein R₁ is R₄C(O)—. Reaction of C2 with the nitrile oxide 1f leads toA8 which is converted to A10 as illustrated in Method 1.

EXAMPLE 7 Compound No. 239

The resin M1K (0.47 mmol) was shaken in 20% piperidine/DMF for 10minutes and then filtered and washed with DMF and DCM. The resultingresin was shaken again overnight with a solution of Cbz-tBG-OH (374 mg,3.0 eq.), HOBT (2.82 mL of 0.5 M in DMF, 3.0 eq.), HBTU (2.82 of 0.5 Min DMF, 3.0 eq.), and DIEA (0.493 mL, 6.0 eq.). The resin was thenfiltered and washed with DMF and DCM to give the resin compound M3A(0.47 g), which was used in next reaction without further purification.

The Cbz resin M3A (0.0611 mmol) in THF was shaken with 3-bromo-phenyloxime (10 eq.) and bleach (5% NaOH) (20 eq.) for 12 hours. The resin wasthen filtered and washed with water, DMF, DCM to give the resin M3B.

The resin M3B was shaken with 95% TFA in water for 30 minutes and theresulting solution was concentrated in vacuo to give the compound M3C(0.031 mmol), (M+1) 740, which was used in next reaction without furtherpurification.

A solution of the compound M3C (0.031 mmol) in DCM (3 mL) was stirredwith Dess-Martin Periodinane (26 mg, 2 eq.) and t-BuOH (26 uL). Afterstiffing for 1 hour, sodium thiosulfate was added to above mixture. Theproduct was extracted with EtOAc and the combined organic layer was thenwashed with water, NaHCO₃, brine and concentrated in vacuo and purifiedby Gilson Prep HPLC to afford Compound No. 239. (M+1) 738.

EXAMPLE 8 Compound No. 535

Compound M1E (10.0 g, 1.0 eq.) was shaken in 20% piperidine/DMF for 10minutes. The resin was filtered and washed with DMF (thrice) followed byDCM (thrice). This process was repeated. The resulting resin was shakenovernight with a solution of(S)-2,3-dimethyl-2-(((S)-tetrahydrofuran-3-yloxy)carbonylamino)butanoicacid (3.46 g, 3.0 eq.), HBTU (28.2 mL of 0.5 M in DMF, 3.0 eq.), HOBt(14.1 mL of 1.0M in DMF, 3.0 eq.), and DIEA (4.91 mL, 6.0 eq.) in DMF.The resin was then filtered and washed with DMF (thrice) and DCM(thrice) to give compound M3E. (M+H=523.1)

Compound M3E (300 mg, 1.0 eq.) was stirred in THF and2-nitro-1-phenylethanone (272 mg, 10.0 eq.) was added to the mixturefollowed by phenyl isocyanate (179 uL, 10.0 eq.) and catalytic TEA (10uL). The resulting mixture was shaken overnight, drained, and washedwith DMF, THF, and DCM (thrice each) to give compound M3F (M+H=669.8).

Compound M3F (0.4 g, 1.0 eq.) was stirred in 1/1 TFA/DCM for 2 hours.The resin was drained and washed with DCM (thrice), all organics wereconcentrated, and DCM was added followed by Dess-Martin Periodinane (97mg, 3.0 eq.). The resulting mixture was stirred for 1 hour, 1 N Na₂S₂O₃was added and again, stirred. The reaction mixture was purified viasilica gel chromatography (10-90% ethyl acetate/hexanes gradient) toyield Compound No. 535. M+H=668.1. ¹H-NMR (500 MHz, CDCl₃): 8.19 (d,2H), 7.61 (t, 1H), 7.47 (t, 2H), 7.19 (d, 1H), 6.93 (d, 1H), 5.52 (d,1H), 5.37-5.33 (m, 1H, 5.24 (s, 1H), 4.78 (t, 1H), 4.32-4.29 (m, 2H),3.93-3.79 (m, 4H), 3.70 (d, 1H), 3.48-3.36 (m, 2H), 2.79 (td, 1H),2.68-2.63 (m, 1H), 2.55-2.50 (m, 1H), 2.12-2.04 (m, 1H), 1.96-1.89 (m,1H), 1.66-1.59 (m, 1H), 1.47-1.37 (m, 2H), 1.00 (s, 9H), 0.94-0.81 (m,6H), 0.63-0.57 (m, 2H).

Listed below in Table 3 are additional compounds of Formula I preparedby Method 3.

TABLE 3 Additional Compounds of Formula I Produced by Method 3. Com-pound Starting Starting Starting No. Material for P¹ Material for C¹Material for R₃ 4 N-((S)- N/A 3-Chloro-5- tetrahydrofuran-3-fluorobenzaldoxime yloxy)carbonyl)-L- tert-butylglycine 8 N-((S)- N/A3-(Cyclopentyloxy)-4- tetrahydrofuran-3- methoxybenzaldehydeyloxy)carbonyl)-L- tert-butylglycine 9 N-((S)- N/A 3,5-Di(trifluoro-tetrahydrofuran-3- methyl)benzaldoxime yloxy)carbonyl)-L-tert-butylglycine 11 N/A (S)-2,5- 3-fluoro-4- dioxopyrrolidin-1-methylbenzaldoxime yl tetrahydrofuran- 3-yl carbonate 15 N-((S)- N/A3-Chloro-4- tetrahydrofuran-3- methoxybenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 16 N—FMOC-L-tert- 2-(tetrahydro-2H- 4- butylglycinepyran-4-yl)acetic Methoxybenzaldoxime acid 25 N-((S)- N/A 3,5-tetrahydrofuran-3- Difluorobenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 32 N-((S)- N/A 3,4- tetrahydrofuran-3-Dichlorobenzaldoxime yloxy)carbonyl)-L- tert-butylglycine 36 N-((S)- N/A3,4- tetrahydrofuran-3- Dimethylbenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 47 N/A (S)-2,5- 4-Ethylbenzaldoxime dioxopyrrolidin-1-yl tetrahydrofuran- 3-yl carbonate 52 N-((S)- N/A 4-Trifluoro-tetrahydrofuran-3- methylbenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 55 N—FMOC-L-tert- 2-cyclohexylacetic4-Chlorobenzaldoxime butylglycine acid 56 N—FMOC-L-tert-2-cyclohexylacetic 3,5- butylglycine acid Dichlorobenzaldoxime 64N-((S)- N/A 4-Trifluoro- tetrahydrofuran-3- methylbenzaldoximeyloxy)carbonyl)-L- tert-butylglycine 66 N—FMOC-L-tert-2-cyclohexylacetic 3-Chloro-4- butylglycine acid fluorobenzaldoxime 70N-((S)- N/A Cyclopentane- tetrahydrofuran-3- carboxaldehydeyloxy)carbonyl)-L- tert-butylglycine 78 N—FMOC-L-tert- 2-(tetrahydro-2H-3,4- butylglycine pyran-4-yl)acetic Dichlorobenzaldoxime acid 82N—FMOC-L-tert- 2-(tetrahydro-2H- Piperonal oxime butylglycinepyran-4-yl)acetic acid 83 N-((S)- N/A 3-Chlorobenzaldoximetetrahydrofuran-3- yloxy)carbonyl)-L- tert-butylglycine 95N—FMOC-L-tert- 2-cyclohexylacetic 3-Chloro-5- butylglycine acidfluorobenzaldoxime 106 N-((S)- N/A 3,5- tetrahydrofuran-3-Difluorobenzaldoxime yloxy)carbonyl)-L- tert-butylglycine 109 N-((S)-N/A 3-Methyl-4- tetrahydrofuran-3- chlorobenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 142 N-((S)- N/A Cyclohexane- tetrahydrofuran-3-carboxaldehyde yloxy)carbonyl)-L- tert-butylglycine 149 N-((S)- N/A3-Trifluoro- tetrahydrofuran-3- methoxybenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 150 N-((S)- N/A 2,2- tetrahydrofuran-3-Dimethylchromane-6- yloxy)carbonyl)-L- carbaldehyde tert-butylglycine171 N—FMOC-L-tert- 2-(tetrahydro-2H- 3-Cyanobenzaldoxime butylglycinepyran-4-yl)acetic acid 177 N—FMOC-L-tert- 2-(tetrahydro-2H-4-Cyanobenzaldoxime butylglycine pyran-4-yl)acetic acid 191 N-((S)- N/A3,5-Dimethyl-4- tetrahydrofuran-3- methoxybenzaldoximeyloxy)carbonyl)-L- tert-butylglycine 196 N-((S)- N/A 3,4-Dimethoxy-tetrahydrofuran-3- benzaldoxime yloxy)carbonyl)-L- tert-butylglycine 198N/A (S)-2,5- 3,5-Dimethyl-4- dioxopyrrolidin-1- methoxybenzaldoxime yltetrahydrofuran- 3-yl carbonate 215 N-((S)- N/A 3,4,5-tetrahydrofuran-3- Trifluorobenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 222 N/A (S)-2,5- 2,2-Difluoro-1,3- dioxopyrrolidin-1-benzodioxole-5- yl tetrahydrofuran- carboxaldehyde 3-yl carbonate 224N-((S)- N/A 3,5-Dimethyl-4- tetrahydrofuran-3- methoxybenzaldoximeyloxy)carbonyl)-L- tert-butylglycine 229 N-((S)- N/A Methyl4-nitrobutyrate tetrahydrofuran-3- yloxy)carbonyl)-L- tert-butylglycine234 N—FMOC-L-tert- 2-(tetrahydro-2H- 3-Chloro-5- butylglycinepyran-4-yl)acetic fluorobenzaldoxime acid 236 N-((S)- N/A 3-Chloro-4-tetrahydrofuran-3- methoxybenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 239 N—CBZ-L-tert- N/A 3-Bromobenzaldoxime butylglycine240 N/A (S)- 4-Trifluoro- tetrahydrofuran-3- methylbenzaldoximeyl-carbonate 244 N-((S)- N/A 3-Trifluoro- tetrahydrofuran-3-methoxybenzaldoxime yloxy)carbonyl)-L- tert-butylglycine 251 N-((S)- N/APhenylnitroethane tetrahydrofuran-3- yloxy)carbonyl)-L-tert-butylglycine 257 N-((S)- N/A 3-Phenylbenzaldoximetetrahydrofuran-3- yloxy)carbonyl)-L- tert-butylglycine 258 N-((S)- N/A3-fluoro-5- tetrahydrofuran-3- trifluoro- yloxy)carbonyl)-L-methylbenzaldoxime tert-butylglycine 270 N—FMOC-L-tert-2-(tetrahydro-2H- 3-Chloro-4- butylglycine pyran-4-yl)aceticfluorobenzaldoxime acid 274 N/A (S)- 3,5- tetrahydrofuran-3-Dichlorobenzaldoxime yl-carbonate 279 N—FMOC-L-tert- 2-(tetrahydro-2H-3-Chloro-4- butylglycine pyran-4-yl)acetic methoxybenzaldoxime acid 285N—FMOC-L-tert- 2-(tetrahydro-2H- 3,5- butylglycine pyran-4-yl)aceticDichlorobenzaldoxime acid 299 N—FMOC-L-tert- 2-(tetrahydro-2H-4-Chlorobenzaldoxime butylglycine pyran-4-yl)acetic acid 301 N-((S)- N/A3-Chloro-4- tetrahydrofuran-3- fluorobenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 306 N-((S)- N/A 3,5- tetrahydrofuran-3-Dichlorobenzaldoxime yloxy)carbonyl)-L- tert-butylglycine 314 N-((S)-N/A Methyl 4- tetrahydrofuran-3- formylbenzoate yloxy)carbonyl)-L-tert-butylglycine 316 N-((S)- N/A 2,2-Difluoro-1,3- tetrahydrofuran-3-benzodioxole-5- yloxy)carbonyl)-L- carboxaldehyde tert-butylglycine 318N-((S)- N/A 4-Chlorobenzaldoxime tetrahydrofuran-3- yloxy)carbonyl)-L-tert-butylglycine 322 N-((S)- N/A 3-Chloro-5- tetrahydrofuran-3-fluorobenzaldoxime yloxy)carbonyl)-L- tert-butylglycine 323N—FMOC-L-tert- 2-cyclohexylacetic 3,4- butylglycine acidDichlorobenzaldoxime 330 N-((S)- N/A 3,5-Di(trifluoro-tetrahydrofuran-3- methyl)benzaldoxime yloxy)carbonyl)-L-tert-butylglycine 348 N/A (S)- 3-fluoro-5- tetrahydrofuran-3- trifluoro-yl-carbonate methylbenzaldoxime 353 N-((S)- N/A Methyl 3-tetrahydrofuran-3- formylbenzoate yloxy)carbonyl)-L- tert-butylglycine362 N—FMOC-L-tert- 2-cyclohexylacetic 3,5-Dimethyl-4- butylglycine acidmethoxybenzaldoxime 363 N-((S)- N/A 2,2-Difluoro-1,3- tetrahydrofuran-3-benzodioxole-5- yloxy)carbonyl)-L- carboxaldehyde tert-butylglycine 364N-((S)- N/A 4- tetrahydrofuran-3- Chlorophenylglyoxyloyloxy)carbonyl)-L- hydroxamyl chloride tert-butylglycine 385 N-((S)- N/A4-Methylbenzaldoxime tetrahydrofuran-3- yloxy)carbonyl)-L-tert-butylglycine 391 N-((S)- N/A 3,5- tetrahydrofuran-3-Dichlorobenzaldoxime yloxy)carbonyl)-L- tert-butylglycine 403 N-((S)-N/A 2-Chloro-6- tetrahydrofuran-3- fluorobenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 405 N/A (S)- 4- tetrahydrofuran-3-Isopropylbenzaldoxime yl-carbonate 413 N/A (S)- 3-Methyl-4-tetrahydrofuran-3- fluorobenzaldoxime yl-carbonate 414 N/A (S)- 3,4,5-tetrahydrofuran-3- Trifluorobenzaldoxime yl-carbonate 423 N-((S)- N/A3-fluoro-4- tetrahydrofuran-3- methylbenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 425 N-((S)- N/A 3-(4- tetrahydrofuran-3-Pyridyl)benzaldehyde yloxy)carbonyl)-L- tert-butylglycine 434N—CBZ-L-tert- N/A 2,3-Dimethoxy- butylglycine benzaldoxime 436N—FMOC-L-tert- 2-cyclohexylacetic 3-Methyl-4- butylglycine acidchlorobenzaldoxime 444 N-((S)- N/A 3-Methylbenzaldoximetetrahydrofuran-3- yloxy)carbonyl)-L- tert-butylglycine 448 N-((S)- N/A3-(4-chlorophenyl(- tetrahydrofuran-3- 2,1-benzisoxazole-5-yloxy)carbonyl)-L- carbaldehydeoxime tert-butylglycine 451 N/A (S)-3-Trifluoromethyl-4- tetrahydrofuran-3- fluorobenzaldoxime yl-carbonate455 N-((S)- N/A 3,4,5- tetrahydrofuran-3- Trifluorobenzaldoximeyloxy)carbonyl)-L- tert-butylglycine 456 N-((S)- N/A 1,4-benzodioxan-6-tetrahydrofuran-3- carboxaldehyde yloxy)carbonyl)-L- tert-butylglycine472 N-((S)- N/A 2-Furanaldoxime tetrahydrofuran-3- yloxy)carbonyl)-L-tert-butylglycine 480 N-((S)- N/A Methyl 3- tetrahydrofuran-3-formylbenzoate yloxy)carbonyl)-L- tert-butylglycine 481 N-((S)- N/A3-(Carboxy)benzal- tetrahydrofuran-3- doxime yloxy)carbonyl)-L-tert-butylglycine 482 N-((S)- N/A 2-Chloro-6- tetrahydrofuran-3-fluorobenzaldoxime yloxy)carbonyl)-L- tert-butylglycine 486N—FMOC-L-tert- 2-cyclohexylacetic 3-Chloro-4- butylglycine acidmethoxybenzaldoxime 490 N—FMOC-L-tert- 2-(tetrahydro-2H- 3-Methyl-4-butylglycine pyran-4-yl)acetic chlorobenzaldoxime acid 498 N-((S)- N/A3-fluoro-5- tetrahydrofuran-3- trifluoro- yloxy)carbonyl)-L-methylbenzaldoxime tert-butylglycine 509 N-((S)- N/A 3-Trifluoro-tetrahydrofuran-3- methylbenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 511 N-((S)- N/A 4-Nitrobenzaldoxime tetrahydrofuran-3-yloxy)carbonyl)-L- tert-butylglycine 519 N-((S)- N/A 3,5-tetrahydrofuran-3- Dimethylbenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 524 N/A (S)- 4-Trifluoro- tetrahydrofuran-3-methoxybenzaldoxime yl-carbonate 525 N—FMOC-L-tert- 2-(tetrahydro-2H- 3-butylglycine pyran-4-yl)acetic Methoxybenzaldoxime acid 530 N-((S)- N/A4- tetrahydrofuran-3- Hydroxybenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 535 N-((S)- N/A 2-nitro-1-phenyl tetrahydrofuran-3-ethanone yloxy)carbonyl)-L- tert-butylglycine 539 N—FMOC-L-tert-2-(tetrahydro-2H- 3-Methyl-4- butylglycine pyran-4-yl)aceticfluorobenzaldoxime acid 543 N-((S)- N/A 4-(Carboxy)benzal-tetrahydrofuran-3- doxime yloxy)carbonyl)-L- tert-butylglycine 545N-((S)- N/A 3-Trifluoromethyl-4- tetrahydrofuran-3- fluorobenzaldoximeyloxy)carbonyl)-L- tert-butylglycine 548 N—FMOC-L-tert-2-cyclohexylacetic 3-Chloro-4- butylglycine acid fluorobenzaldoxime 550N-((S)- N/A 3-fluoro-4- tetrahydrofuran-3- methylbenzaldoximeyloxy)carbonyl)-L- tert-butylglycine 551 N-((S)- N/A Methyl 4-tetrahydrofuran-3- formylbenzoate yloxy)carbonyl)-L- tert-butylglycine555 N-((S)- N/A 3-Methyl-4- tetrahydrofuran-3- fluorobenzaldoximeyloxy)carbonyl)-L- tert-butylglycine 560 N-((S)- N/A 3-Trifluoro-tetrahydrofuran-3- methylbenzaldoxime yloxy)carbonyl)-L-tert-butylglycine 572 N—FMOC-L-tert- 2-(tetrahydro-2H- 3,5-Dimethyl-4-butylglycine pyran-4-yl)acetic methoxybenzaldoxime acid

Certain other compounds of Formula I may be prepared as illustrated byMethod 4.

Method 4:

Referring to Method 4, the Fmoc derivative A3 is prepared as describedin Method 1. Reaction of A3 with the resin bound imino amide D1 in thepresence of a coupling reagent provides the compound bound resin D2. Theresin bound imino amide D1 may be prepared from the diketo compound X31by reaction with an amino resin such as, for example, a derivatizedaminomethylated polystyrene, e.g., X32. Deprotection of D2 provides D3which reacts with an R₁ carboxylic acid in the presence of a couplingreagent to provide D4 wherein R₁ is R₄C(O)—. Reaction of D4 with thenitrile oxide if provides D5 which on hydrolysis from the resin providesA10.

EXAMPLE 9 Compound No. 303

To a suspension of resin M4A, which has the same structure as X33, (20g, 0.4 mmol/g, 8 mmol) in DCM (100 mL) was added PPh₃ (21 g, 80 mmol),dimethyl barbituric acid (12.5 g, 80 mmol) and Pd(PPh₃)₄ (920 mg, 0.8mmol). The suspension was shaken overnight, drained, washed with DMF (10times) and DCM (4 times). N-Fmoc-4-methyleneproline (3.0 g, 8.8 mmol),HBTU (3.3 g, 8.8 mml) and HOBt (1.1 g, 8.8 mmol) and DIEA (1.6 mL, 8.8mmol) were dissolved in DMF (100 mL). The solution was added to theresin and shaken overnight. The resin was then drained, washed with DMF(10 times), DCM (4 times) and dried to afford resin M4B.

To the resin M4B (20 g, 8 mmol) was added 20% piperdine in DMF (100 mL),shaken for 1 hour, and then washed with DMF (10 times), DCM (4 times).To the resin was added a mixture of Fmoc-tert-butylglycine (5.6 g, 16mmol), HBTU (6.1 g, 16 mmol), HOBt (2.2 g, 16 mmol) and (iPr)₂NEt (2.9mL, 16 mmol) in DMF (100 mL). The suspension was shaken overnight,drained, washes with DMF (10 times), DCM (4 times) and dried to affordthe resin M4C.

To the resin M4C (20 g, 8 mmol) was added 20% piperdine in DMF (100 mL),shaken for 1 hour, and then washed with DMF (10 times), DCM (4 times).To the resin was added a mixture of cyclohexylacetic acid (1.42 g, 10mmol), HBTU (3.8 g, 10 mmol), HOBt (1.35 g, 10 mmol) and (iPr)₂NEt (1.8mL, 10 mmol) in DMF (100 mL). The suspension was shaken overnight,drained, washes with DMF (10 times), DCM (4 times) and dried to affordthe resin M4D.

A solution of 3-pyridinealdoxime (M4E) (122 mg, 1 mmol) in DMF (3 mL)was added NCS (134 mg, 1 mmol). The mixture was heated to 50-60° C. for30 minutes. After cooling down to room temperature, the3-pyridinechloroxime (M4F) solution was added to a resin M4D (300 mg,0.12 mmol). To the mixture was added TEA (0.14 mL, 1 mmol) and thereaction mixture was heated to 50-60° C. for 4 hours. The reactionmixture was drained and washed with DMF (6 times) and DCM (6 times). Theresin was treated with 95% TFA for 5 hours. The mixture was drained, andwashed with DCM. The filtrate was concentrated in vacuo, purified fromcolumn 50-100% EtOAc/Hex to afford 7 mg colorless solid as productCompound No. 303. HPLC 5.7-6.4 minutes; MS 651.5 and LC-MS 3.9 minutes.

Listed below in Table 4 are additional compounds produced by Method 4.

TABLE 4 Additional Compounds of Formula I Produced by Method 4. Com-pound Starting Starting Starting No. Material for P¹ Material for C¹Material for R₃ 41 N—FMOC-L- 2-Cyclohexyl- 5-Bromoprydine-3-tert-butylglycine acetic acid carbaldehyde 179 N—FMOC-L- 2-Cyclohexyl-5-Bromo-2-Furaldehyde tert-butylglycine acetic acid 230 N—FMOC-L-2-Cyclohexyl- 2-methylbenzofuran-3- tert-butylglycine acetic acidcarbaldehyde 303 N—FMOC-L- 2-Cyclohexyl- 3-Pyridine-carboxaldehydetert-butylglycine acetic acid 495 N—FMOC-L- 2-Cyclohexyl-4-Chloro-1-methyl-1H- tert-butylglycine acetic acidpyrazole-3-carbaldehyde 552 N—FMOC-L- 2-Cyclohexyl-2,3-Dihydrobenzo[b]furan-5- tert-butylglycine acetic acid carboxaldehyde

Certain other compounds of the invention may be prepared as illustratedin Methods 5a and 5b.

II. Method 5a

Referring to Method 5a, the exomethylene acid compound A1 is protectedto provide the di-t-butyl dicarboxylate E1. Reaction of E1 with nitrileoxide of formula 1f provides the intermediate B1 which is transformedinto amino acid derivative E2. Reaction of E2 with an aminoalcohol E5provides A9. Compound A9 is converted to A10 as described in Method 1.

III. Method 5b

Referring to Method 5b, the intermediate compound B1 is transformed intoamino acid ester E6. Reaction of E6 with an R₁ carboxylic acid in thepresence of a coupling reagent provides E7 wherein R₁ is R₄C(O)—. E7 isdeprotected to provide E2 which is converted to A10 as described inMethod 1.

EXAMPLE 10 Compound No. 422

Compound 10A (5.0 g, 1.0 eq.) was stirred in 100 mL acetonitrile and tothe solution were added ditertbutyldicarbonate (9.6 g, 2 eq.),dimethylaminopyridine (537 mg, 0.2 eq.), and triethylamine (6.13 mL, 2.0eq.). The mixture was stirred overnight, concentrated, added ethylacetate, washed with 1.0N HCl, dried over sodium sulfate, concentrated,and purified with silica gel chromatography (10-30% ethylacetate/hexanes gradient) to give compound 10B (80%). (M+H=284.0).¹H-NMR (CDCl₃): 5.0 (m, 2H), 4.3-4.5 (m, 1H), 4.0-4.1 (m, 2H), 2.9-3.0(m, 1H), 2.5-2.6 (d, 1H), 1.5(s, 3/9 of 18H), 1.4(s, 6/9 of 18H).

Compound 10B (10.0 g, 1.0 eq.) was stirred in 175 mL DCM withpiperonaloxime (11.5 g, 2.0 eq.). The solution was cooled on an ice bathand to it added bleach (175 mL) slowly. The mixture was then warmed toroom temperature, stirred for 2 hours, separated and its aqueous layerextracted with DCM twice. Organics were combined and dried overmagnesium sulfate, filtered and concentrated. The residue was purifiedand separated diastereomers by silica gel chromatography (5-30% ethylacetate/hexanes gradient) to yield 4.1 g of Compound 10C (26%).(M+H=446.9.) ¹H-NMR (CDCl₃): 7.25 (m, 1H), 7.0 (d, 1H), 6.8 (d, 1H), 6.0(s, 2H), 4.6-4.4 (m, 1H), 4.0-3.8 (m, 1H), 3.7-3.6 (m, 1H), 3.4-3.3 (m,1H), 3.3-3.2 (m, 1H), 2.8-2.7 (m, 1H), 2.3-2.2 (m, 1H), 1.5 (s, 9H), 1.4(s, 9H).

Alternatively, compound 10B was prepared by the following procedures:

Preparation: (S)-di-tert-butyl 4-methylenepyrrolidine-1,2-dicarboxylate

Procedure 1

Triethylamine (2 eq.) was added to a solution of(S)-1-(tert-butoxycarbonyl)-4-methylenepyrrolidine-2-carboxylic acid(1.0 eq.), di-tert-butyldicarbonate (2.0 eq.), and DMAP (0.2 eq.) inacetonitrile (10 vol) at ambient temperature. The reaction mixture wasstirred for 16 h, then diluted with isopropyl acetate (25 vol). A washwith water (20 vol., twice) was followed by a filtration over Na₂SO₄ andsolvent removal. The crude product was purified by filtration through apad of silica gel (37 vol silica, first flush with heptane (80 vol),second flush with 10% ethyl acetate in heptane (30 vol)). Removal ofsolvent from the second flush gave compound 10B.

Procedure 2

A solution of di-tert-butyl dicarbonate (1.1 eq.) in MTBE (2 vol.) wasadded to a mixture of(S)-1-(tert-butoxycarbonyl)-4-methylenepyrrolidine-2-carboxylic acid(1.0 eq.) and DMAP (0.2 eq.) in MTBE (8 vol) and t-butanol (1.75 vol.).The mixture was stirred for 1 hour, at which point gas evolution ceased.The mixture was washed with 1 N HCl (3 vol.), then saturated aqueousNaHCO₃ (3 vol.) and then brine (3 vol.). The solvent is then removed toafford compound 10B.

Compound 10C (4.0 g, 1.0 eq.) was stirred in 1/1 TFA/DCM for 3 hours andthe solution was concentrated. To the concentrate was added 100 mLacetone, 100 mL saturated sodium bicarbonate solution, andditertbutyldicarbonate and the resulting solution was stirred overnightand then acidified with 1.0 N HCl solution and extracted with ethylacetate (thrice). The organics were washed with brine solution and driedover magnesium sulfate, filtered and concentrated to yield 4.0 g ofCompound 10D (M+H=391.1).

Compound 10D (50 mg, 1.0 eq.) stirred in 0.5 mL DMF with EDC (37 mg, 1.5eq.), PS-HOBt (137 mg, 1.5 eq.) and NMM (56 uL, 4.0 eq.), and to thesolution was added 0.5 mL DCM to assist in swelling of the resin. To themixture was added 3-amino-2-hydroxyhexanamide (30 mg, 1.3 eq.) and themixture was stirred overnight, filtered, diluted with ethyl acetate,washed with 1.0 N HCl, dried over sodium sulfate, filtered, andconcentrated. The solution was purified by silica gel chromatography(100% DCM-5% MeOH/DCM gradient) to yield 21 mg of the crude product,which was then stirred in 4.0 N HCl/dioxane for 2 hours and concentratedto yield compound 10E as an HCl salt (M+H=419.0).

Compound 10E (21 mg, 1.0 eq.) was stirred in DMF with NMM (13 uL, 1.4eq.) and to the solution was added a solution of(S)-3,3-dimethyl-2-(((S)-tetrahydrofuran-3-yloxy)carbonylamino)butanoicacid (14 mg, 1.4 eq.), EDC (11 mg, 1.4 eq.), and PS-HOBt (40 mg, 1.4eq.) in DMF, with enough DCM to swell the resin. The mixture was stirredovernight, filtered, washed with 1.0 N HCl, dried over sodium sulfate,filtered and then concentrated to give compound 10F, which was usedwithout further purification. (M+H=646.4)

Compound 10F was stirred in DCM and to the solution was addedDess-Martin Periodinane (˜3.0 eq.). The solution was stirred for 1 hour,added to it 1.0 N Na₂S₂O₃, and stirred. The mixture was purified bysilica gel chromatography (10-90% ethyl acetate/hexanes gradient) toyield 9 mg of Compound No. 422 (M+H=644.3). ¹H-NMR (CDCl₃): 7.3 (m, 1H),7.15(m, 1H), 6.95 (m, 1H), 6.8 (m, 1H), 6.75 (m, 1H), 6.0 (s, 2H),5.5-5.4 (m, 2H), 5.4-5.3 (m, 2H), 4.8-4.7 (m, 1H), 4.3 (m, 1H), 4.2 (m,1H), 4.0-3.8 (m, 3H), 3.7 (m, 1H), 3.4-3.2 (m, 2H), 2.6 (m, 1H,), 2.5(m, 1H), 2.2-2.1 (m, 1H), 2.1-2.0 (m, 1H), 1.9 (m, 1H), 1.6 (m, 1H),1.5-1.4 (m, 2H), 1.0-0.9 (m, 13H).

EXAMPLE 11 Compound No. 562

To 2,4-dimethoxybenzaldoxime (4.5 g, 24.8 mmol) in DMF (135 mL) wasadded dropwise over 2 h at room temperature a solution ofN-chlorosuccinimide (6.6 g, 49.7 mmol) in DMF (135 mL). The reaction wasstirred 14 hours and compound 11A (5.2 g, 18.4 mmol) was added followedby dropwise addition over 1 h of a solution of triethylamine in DMF (2.6mL, 18.4 mmol, in 15 mL). After stirring for 3 h, the reaction mixturewas washed with H₂O and dried over MgSO₄. The resulting residue waspurified via silica gel chromatography to afford 5.8 g (63%) of compound11B as a tan solid. ES (+) MS: m/e 497 (M+H)⁺.

To compound 11B (5.5 g, 11.1 mmol) in CH₂Cl₂ (30 mL) was addedtrifluoroacetic acid (30 mL). The reaction mixture was stirred for 90minutes at room temperature and concentrated under reduced pressure toprovide a tan solid, which was dissolved in MeOH (60 mL) and heated toreflux. Concentrated sulfuric acid (˜5 mL) was added dropwise and thereaction was refluxed for 3 hours, after which the solvent was removedunder reduced pressure. The resulting residue was dissolved in CH₂Cl₂(75 mL) and carefully treated with a saturated NaHCO₃ solution untilpH˜9. The organic layer was dried over MgSO₄ and concentrated to providethe intermediate amino ester. To N-Boc-tert-butylglycine (3.1 g, 13.6mmol) in CH₂Cl₂ (60 mL) was added EDC (2.6 g, 13.6 mmol), HOBt (1.8 g,13.6 mmol) and triethylamine (5.5 mL, 39.5 mmol). After stirring 5minutes, the above amino ester was added and the reaction was stirred atroom temperature 14 hours. The reaction mixture was washed with H₂O, 1 NHCl, and saturated NaHCO₃ solution. The organic layer was dried overMgSO₄ and concentrated in vacuo to provide 5.6 g of compound 11C (87%over 3 steps) as a brown solid which was used without furtherpurification. ES (+) MS: m/e 568 (M+H)⁺.

To compound 11C (600 mg, 1.1 mmol) in CH₂Cl₂ (3 mL) was addedtrifluoroacetic acid (3 mL). The reaction was stirred for 1 hour andconcentrated under reduced pressure to give the desired amine product asthe TFA salt. To cyclohexylacetic acid (181 mg, 1.3 mmol) in CH₂Cl₂ (6mL) was added EDC (243 mg, 1.3 mmol), HOBt (171 mg, 1.3 mmol) andtriethylamine (516 μL, 3.7 mmol). After stirring for 5 minutes, theabove amine was added and the reaction was stirred at room temperature14 hours. The reaction mixture was washed with H₂O, 1 N HCl, andsaturated NaHCO₃ solution. The organic layer was dried over MgSO₄ andconcentrated in vacuo, and the resulting residue was purified via silicagel chromatography to provide 460 mg of compound 11D (74% over 2 steps)as an off-white solid. ES (+) MS: m/e 592 (M+H)⁺.

To compound 11D (460 mg, 0.8 mmol) in a solution of THF/H₂O (5 mL, 3:1v/v) was added LiOH monohydrate (82 mg, 1.9 mmol). The reaction mixturewas stirred at room temperature 14 hours, acidified using 1 N HCl, andextracted with EtOAc. The organic layer was dried over MgSO₄ andconcentrated under reduced pressure to provide 405 mg of compound 11E,which was used without further purification. ES (+) MS: m/e 578 (M+H)⁺.

To compound 11E (80 mg, 0.14 mmol) in CH₂Cl₂ (1 mL) was added EDC (38mg, 0.2 mmol), HOBt (27 mg, 0.2 mmol) and triethylamine (68 μL, 0.5mmol). After stirring for 5 minutes, compound 11F was added and thereaction was stirred at room temperature 14 hours. The reaction mixturewas washed with H₂O, 1 N HCl, and saturated NaHCO₃ solution. The organiclayer was dried over MgSO₄ and concentrated in vacuo to provide 95 mg ofcompound 11G (95%) as a brown solid which was used without furtherpurification. ES (+) MS: m/e 718 (M+H)⁺.

To compound 11G (95 mg, 0.14 mmol) in CH₂Cl₂ (1 mL) was addedDess-Martin periodinane (71 mg, 0.17 mmol). After stirring for 30minutes, the reaction was quenched with 1 N Na₂S₂O₃. The organic layerwas purified via silica gel chromatography to give Compound No. 562,i.e., compound 11H shown above, as a white solid. ES (+) MS: m/e 716(M+H)⁺.

EXAMPLE 12 Compound No. 362

4-Methoxy-3,5-dimethylbenzaldehyde (1.86 g, 11.3 mmol) was dissolved inethanol (30 mL) and stirred with hydroxylamine hydrochloride (2.4 M aq.solution, 5.65 mL, 1.2 eq.) and Na₂CO₃ (1.2 M solution, 5.65 mL, 0.6eq.) at room temperature for 2.5 hours. The mixture was then heated to60° C. and additional hydroxylamine hydrochloride and Na₂CO₃ was added.The mixture was again stirred overnight at 60° C., transferred to aseparatory funnel, diluted with EtOAc. The organic layer was separated,washed with brine, dried over MgSO₄, filtered and concentrated. Theproduct was purified by ISCO chromatography with EtOAc/hexanes eluent toyield 1.55 g (8.56 mmol, 77%) of 4-methoxy-3,5-dimethylbenzaldehydeoxime as a white solid. M+1=180.0.

To a solution 4-methoxy-3,5-dimethylbenzaldehyde oxime (1.34 g, 7.48mmol) in DMF (10 mL) was added N-chlorosuccinimide (1.76 g, 13.2 mmol).This solution was stirred until starting material was consumed asindicated by HPLC. To the solution was then added a solution of(S)-di-tert-butyl 4-methylenepyrrolidine-1,2-dicarboxylate (2.1 g, 1.0eq.) in DMF (5 mL). To the solution was added triethylamine (1.2 eq.)dropwise, and the reaction mixture was stirred for 2 hours. The reactionwas then diluted with EtOAc and the organic phase was washed with water,brine, dried (MgSO₄), filtered and concentrated. The product waspurified over silica gel on an ISCO Combiflash using EtOAc/hexanes asthe eluent to yield 912 mg (1.98 mmol) of compound 12A. M+1=461.4.¹H-NMR (500 MHz, CDCl₃): 7.30 (s, 2H), 4.40-4.32 (m, 1H), 3.98-3.79 (m,1H), 3.74 (s, 3H), 3.64-3.58 (m, 1H), 3.40-3.34 (m, 1H), 3.24-3.19 (m,1H), 2.72 (dd, J=8.7, 12.9 Hz, 1H), 2.29 (s, 6H), 2.11-2.07 (m, 1H),1.54-1.45 (m, 18H).

Compound 12A (910 mg, 1.98 mmol) was stirred in CH₂Cl₂/trifluoroaceticacid (1:1, 20 mL) until HPLC indicated complete deprotection of startingmaterial. The intermediate amino acid was concentrated and thendissolved in methanol (30 mL) and heated to reflux with concentratedH₂SO₄ until the starting material was consumed as indicated by HPLC.Concentrated material in vacuo, then dissolved in EtOAc and washed withNaHCO₃, brine, dried over MgSO₄ and concentrated to give compound 12B.M+1=319.0

Compound 12B (727 mg, 2.28 mmol) was dissolved in DMF (3 mL) withBoc-t-butylglycine (686 mg, 3.0 mmol), EDC•HCl (659 mg, 3.43 mmol), HOBt(460 mg, 3.4 mmol), and DIEA (1.2 mL, 6.89 mmol) and stirred at roomtemperature overnight. The reaction was then transferred to a separatoryfunnel and diluted with EtOAc. The organic layer was washed with 1 N HCl(twice, 20 mL each), sat. aq. NaHCO₃ (25 mL), water (10 mL), brine (10mL), dried over MgSO₄ and concentrated. The crude product 12C waspurified over silica gel on an ISCO Combiflash with EtOAc/Hexanes aseluent to yield 231 mg (0.435 mmol) of compound 12C as a clear colorlessoil. LCMS (M+1)=532.45

Compound 12C (231 mg, 0.435 mmol) was stirred in 4N HCl in dioxane (15mL) for 90 minutes at which point TLC analysis indicated no startingmaterial was present in the reaction mixture. The HCl and dioxane wereevaporated to yield an off-white foam. A portion of this intermediate(0.35 mmol), EDC•HCl (96 mg, 0.50 mmol), HOBt (72 mg, 0.53 mmol), andcyclohexaneacetic acid (78 mg, 0.55 mmol) were stirred in DMF (3.5 mL).To this was added DIEA (0.18 mL, 1.0 mmol) and the reaction was stirredovernight. The reaction was then diluted with EtOAc and transferred to aseparatory funnel where the layers were separated and the organic phasewas washed with 1.0 N HCl, saturated aq. NaHCO₃, brine, dried over MgSO₄and concentrated. The product was purified over silica gel on an ISCOCombiflash with EtOAc/hexane as eluent to yield 219 mg (0.394 mmol) ofcompound 12D as a clear oil. M+1=556.4

Compound 12D (219 mg, 0.394 mmol) in THF/H₂O/MeOH (4:1:1, 6 mL) wasstirred with LiOH.H₂O (1.5 eq.) at room temperature overnight. Thereaction was then acidified with 1.0 N HCl and extracted with CH₂Cl₂.The organic layer was washed with brine, dried over MgSO₄ andconcentrated to yield 207 mg (0.382 mmol) of compound 12E. M+1=548.4

Compound 12E (207 mg, 0.382 mmol) was stirred with HOBt (107 mg, 0.792mmol), EDC•HCl (144 mg, 0.764 mmol), and hydroxyamine hydrochloride (168mg, 0.75 mmol) in DMF (2.0 mL) at room temperature and treated with DIEA(0.400 mL, 2.3 mmol). The reaction was stirred overnight, diluted withEtOAc, washed with 1N HCl, saturated NaHCO₃, and the combined aqueouslayers were back extracted with EtOAc. The organic layers were combined,dried over MgSO₄, concentrated and purified over silica gel on an ISCOcombiflash with EtOAc/Hexanes as eluent to yield 227 mg (0.320 mmol) ofcompound 12F as a white solid. (M+TFA) M−1=822.6.

Compound 12F (227 mg, 0.320 mmol) was dissolved at room temperature inCH₂Cl₂ (4 mL) and treated with Dess-Martin periodinane (142 mg, 1.0eq.). After 15 minutes, TLC showed the reaction to be complete, and thereaction solution was quenched by the addition of water and stirredvigorously. Additional CH₂Cl₂ was added, the organic layer was separatedand purified over silical gel on an ISCO combiflash with EtOAc/Hexanesas eluent to yield 159 mg (0.225 mmol) of Compound No. 362. FIA MS(M+1)=708.42. ¹H-NMR (500 MHz, CDCl₃): 7.30 (s, 2H), 7.17 (d, 1H), 6.93(d, 1H), 6.15 (d, 1H), 5.39-5.33 (m, 1H), 4.72 (t, 1H), 4.66 (d, 1H),4.25 (d, 1H), 3.74 (s, 3H), 3.74-3.69 (m, 1H), 3.42 (d, 1H), 3.30 (d,1H), 2.81-2.75 (m, 1H), 2.58-2.46 (m, 2H), 2.29 (s, 6H), 2.16-2.10 (m,1H), 2.08-2.00 (m, 1H), 1.97-1.88 (m, 1H), 1.85-1.57 (m, 8H), 1.51-1.35(m, 2H), 1.33-1.22 (m, 2H), 1.20-1.07 (m, 1H), 1.02-0.96 (m, 10H), 0.92(t, 3H), 0.88-0.80 (m, 2H), 0.66-0.56 (m, 2H).

EXAMPLE 13 Compound No. 247

To a solution of compound 13A (222 mg, 0.5 mmol) was added TEA (0.14 mL)and t-butylisocyanate (0.6 mmol). The resulting solution was stirredovernight and then diluted with EtOAc (20 mL), washed with water (10mL), dried over Na₂SO₄ and concentrated in vacuo. The crude product waspurified chromatography on silica gel to afford compound 13B as a whitesolid (190 mg). HPLC 8.48 min; LC-MS m/z 507.2 ES⁺.

Compound 13B was dissolved in THF and the solution was treated with 1.0N aqueous LiOH and water. The reaction mixture was stirred for 1 hour,and concentrated in vacuo. The residue was then diluted with water,washed with Et₂O and acidified with 1 N aqueous HCl. The resultingmixture was extracted twice with CH₂Cl₂ and the combined organics weredried over MgSO₄, filtered and concentrated in vacuo to give crudecompound 13C which was used without further purification for the nextstep. LC-MS m/z 493.22 ES⁺, 491.21 ES⁻.

A solution of compound 13C (20.6 mg) in CH₂Cl₂ (800 μL) was treated with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10 mg) andhydroxybenzotriazole (7 mg) for 1 hour. Diisopropylamine (16 μL) and3-amino-4-cyclobutyl-2-hydroxybutanamide D (10.5 mg) were then added inone portion and the resulting reaction solution was stirred at roomtemperature for another 16 hours. The mixture was then washed with 1Naqueous HCl, 1:1 solution of 1N aqueous K₂CO₃:1N aqueous NaHCO₃, andbrine in succession. The organics were then dried (MgSO₄), concentratedin vacuo and purified by chromatography over silica (0% to 4% MeOH inCH₂Cl₂) to yield compound 13D (11.6 mg). LC-MS m/z 647.25 ES⁺.

A solution of compound 13D (11.6 mg) in CH₂Cl₂ (1 mL) was charged withDess-Martin periodinane (8.4 mg) and the reaction mixture was stirred atroom temperature for 2 hours. The resulting white mixture was thenwashed with 1.0 N aqueous Na₂S₂O₃, the phase were separated and theorganics were the dried over MgSO₄, concentrated in vacuo and purifiedby chromatography over silica (30% to 65% EtOAc in hexanes) to yield 6.7mg of Compound No. 247 as a white solid: ¹H-NMR (500 MHz, CDCl₃): 7.61(s), 7.52 (d, J=6.1 Hz), 7.39 (d, J=7.8 Hz), 7.34 (t, J=7.8 Hz), 6.87(s), 6.77 (s), 5.89 (s), 5.67 (s), 5.23-5.19 (m), 4.83-4.79 (m), 4.47(s), 4.38 (d, J=11.0 Hz), 3.72 (dd, J=3.1, 11.2 Hz), 3.45 (m), 3.30 (d),2.64 (m), 2.56 (m), 2.44-2.36 (m), 2.08-1.98 (m), 1.86-1.68 (m),1.64-1.58 (m), 1.33-1.22 (m), 1.05-1.00 (m, H), 0.95-0.92 (m, H) ppm.LC-MS m/z 647.25 ES⁺.

EXAMPLE 14 Compound No. 57

A solution of compound 14A (512 mg) in dioxane was treated with 4 N HClin dioxane. The reaction solution was stirred at room temperature for 45minutes and concentrated in vacuo. The resulting residue was dissolvedin a small amount of CH₂Cl₂ and crystallized from Et₂O/Hexanes to givecompound 14B as a white solid (362 mg, 80%). LC-MS m/z 468.24 ES⁺.

A solution of cycloheptane acetic acid (83 mg, Aldrich Chemical Co.,Milwaukee, Wis.) in CH₂Cl₂ (4 mL) was treated with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (103 mg) andhydroxybenzotriazole (72 mg) for 1 hour. Diisopropylamine (160 μL) andintermediate 14B (179 mg) were then added in one portion and theresulting reaction solution was stirred at room temperature for another2 hours. The mixture was then washed with 1 N aqueous HCl, 1:1 solutionof 1 N aqueous K₂CO₃:1 N aqueous NaHCO₃, and brine in succession. Theorganics were the dried (MgSO₄), concentrated in vacuo and purified bychromatography over silica (15% to 60% EtOAc in hexanes) to yieldcompound 14C (188 mg, 88%). LC-MS m/z 606.25 ES⁺.

Compound 14C (186 mg) was dissolved in THF (3 mL) and the solution wastreated with 1 N aqueous LiOH (620 μL) and water (1 mL). The reactionmixture was stirred for 45 minutes at room temperature, and concentratedin vacuo. The residue was then diluted with water, washed with Et₂O andacidified with 1 N aqueous HCl. The resulting mixture was extractedtwice with EtOAc and the combined organics were dried over MgSO₄,filtered and concentrated in vacuo to give crude compound 14D which wasused without further purification for the next step. LC-MS m/z 592.25ES⁺, 590.35 ES⁻.

A solution of compound 14D (89 mg) in CH₂Cl₂ (1 mL)/DMF (1 mL) wastreated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(44 mg) and hydroxybenzotriazole (31 mg) for 1 hour. Diisopropylamine(70 μL) and (3S)-3-amino-4-cyclopropyl-2-hydroxybutanamide (35 mg) werethen added in one portion and the resulting reaction solution wasstirred at room temperature for another 16 hours. The mixture was thenwashed with 1 N HCl, 1:1 solution of 1N aqueous K₂CO₃:1N aqueous NaHCO₃,and brine in succession. The organics were the dried over MgSO₄,concentrated in vacuo and purified by chromatography over silica (0% to5% MeOH in CH₂Cl₂) to yield 96 mg of compound 14F (87%). LC-MS m/z732.21 ES⁺.

A solution of compound 14F (96 mg) in CH₂Cl₂ (1.5 mL) was charged withDess-Martin periodinane (83 mg) and the reaction mixture was stirred atroom temperature for 2 hours. The resulting white mixture was thenwashed with 1 N aqueous Na₂S₂O₃, the phase were separated and theorganics were the dried over MgSO₄, concentrated in vacuo and purifiedby chromatography over silica (10% to 95% EtOAc in hexanes) to yieldCompound No. 57 (44 mg) as a white solid. ¹H-NMR (500 MHz, CDCl₃): 7.76(s), 6.75 (br s), 6.48 (s), 6.07 (d), 5.40 (m), 4.67 (m), 4.22 (d), 3.95(s), 3.87 (s), 3.75 (d), 3.43 (m), 2.51 (m), 2.10 (m), 1.30-1.87 (m),1.12-1.28 (m), 0.97 (m), 0.79 (m), 0.15 (m), 0.03 (m) ppm. LC-MS m/z730.35 ES⁺, 728.35 ES⁻.

EXAMPLE 15 Compound No. 600

Compound 600 has the same structure as compound 266 in Table A.

To a solution of (R)-2-cyclohexylbut-3-ynoic acid (430 mg, 2.4 mmol) inCH₂Cl₂ (10 mL) was added EDC (458 mg, 2.4 mmol), HOBt (324 mg, 2.4 mmol)and triethylamine (836 μL, 6.0 mmol). After stiffing for 5 minutes,compound 15A (800 mg, 2.0 mmol) was added and the reaction was stirred16 hours. The mixture was washed with H₂O, 1 N HCl, and saturated NaHCO₃solution. The organic layer was dried over MgSO₄ and concentrated underreduced pressure to provide 1.23 g crude compound 15B, which waspurified by silica gel chromatography. ES (+) MS: m/e 570 (M+H)⁺.

To a solution of compound 15B (220 mg, 0.4 mmol) in THF/H₂O (2 mL, 3:1v/v) was added LiOH monohydrate (115 mg, 3 mmol). The mixture wasstirred for 2 hours, acidified using 1 N HCl (6 mL) and extracted withEtOAc (thrice, 10 mL). The combined organics were dried over MgSO₄ andconcentrated to afford a colorless oil which was used without furtherpurification. The oil was dissolved in CH₂Cl₂ (2 mL), then EDC (90 mg,0.5 mmol), HOBt (63 mg, 0.5 mmol) and triethylamine (163 μL, 1.2 mmol)were added. After stirring for 5 minutes,(3S)-3-amino-N-cyclopropyl-2-hydroxyhexanamide (87 mg, 0.5 mmol) wasadded. The reaction was stirred 12 hours, washed with H₂O, 1 N HCl, andsaturated NaHCO₃ solution. The organic layer was dried over MgSO₄ andconcentrated under reduced pressure to provide 215 mg of compound 15C asa colorless oil, which was used without further purification. ES (+) MS:m/e 724 (M+H)⁺.

To a solution of compound 15C (53 mg, 0.07 mmol) in CH₂Cl₂ (0.5 mL) wasadded Dess-Martin periodinane (41 mg, 0.1 mmol). The mixture was stirredfor 30 minutes, quenched with 1 Na₂S₂O₃, and separated. The organiclayer was purified by silica gel chromatography to provide 20 mg ofCompound No. 600. ¹H-NMR (500 MHz, CDCl₃): 7.53 (d, J=1.6 Hz, 1H), 7.43(d, J=7.6 Hz, 1H), 7.31-7.25 (m, 2H), 6.83 (d, J=3.3 Hz, 1H), 6.24-6.21(m, 1H), 5.30-5.26 (m, 1H), 4.70-4.58 (m, 2H), 4.23-4.21 (m, 1H), 3.64(dd, 1H), 3.36-3.20 (m, 2H), 2.70-2.68 (m, 1H), 2.57-2.35 (m), 2.04-1.82(m), 1.72-1.30 (m, 10H), 1.18-0.75 (m), 0.55-0.40 (m).

EXAMPLE 16 Compound No. 602

Compound 602 has the same structure as compound 212 in Table A.

To a solution of compound 15C prepared above (20 mg, 0.03 mmol) andazidomethyl pivalate (4 mg, 0.03 mmol, prepared according to Syn. Lett.,2005, 18, pp. 2847-2850) in tert-butanol/H₂O (120 μL, 1:1 v/v) was addedan aqueous solution of sodium ascorbate (10 μL, 0.01 mmol, 1.0 M)followed by an aqueous solution of copper(II) sulfate pentahydrate (5μL, 0.001 mmol, 0.3 M). The reaction mixture was stirred 12 hours atroom temperature, diluted with H₂O, and extracted with EtOAc. Thecombined organics were washed with 5% ammonium hydroxide followed bybrine, and were dried over MgSO₄ and concentrated under reduced pressureto provide 25 mg of crude compound 16B, which was used without furtherpurification. ES (+) MS: m/e 881 (M+H)⁺.

To a solution of compound 16B in MeOH (120 μL) was added aqueous NaOH(120 μL, 1 M). The reaction was stirred at room temperature for 2 hours,then treated with 1 M HCl (120 μL) followed by H₂O (120 μL). The mixturewas extracted with CH₂Cl₂ (thrice, 200 μL each). The combined extractswere washed with brine and concentrated to a volume of approximately 100μL. To this solution was added Dess-Martin periodinane (17 mg, 0.04mmol) and the reaction was stirred 30 minutes. The mixture was quenchedwith 1 M Na₂S₂O₃ (150 μL), and the organic layer was separated andpurified via silica gel chromatography to afford 3 mg of Compound No.602. ES (+) MS: m/e 765 (M+H)⁺.

Listed below in Table 5 are additional compounds of Formula I preparedby Methods 5a and 5b.

TABLE 5 Additional Compounds of Formula I Produced by Methods 5a and 5b.Com- pound Starting Starting Starting No. Material for P¹ Material forC¹ Material for R₃ 5 N—BOC-L-tert- 2-(4-hydroxy-4- 3- butylglycinemethylcyclo- Chlorobenzaldoxime hexyl)acetic acid 10 (S)-4- N/A 3-(benzylamino)-2- chlorobenzaldoxime isopropyl-4- oxobutanoic acid 13N—BOC-L-tert- 2-Norbornaneacetic 3- butylglycine acid chlorobenzaldoxime19 N—BOC-L-tert- 2-(bicy- 3- butylglycine clo[4.1.0]heptan-chlorobenzaldoxime 1-yl)acetic acid 21 (S)-4-(cyclo- N/A 3-hexylamino)-2- chlorobenzaldoxime isopropyl-4- oxobutanoic acid 23N—BOC-L-tert- 2-Cyclohexylacetic 3- butylglycine acid Chlorobenzaldoxime26 N—BOC-L-tert- N-Benzoyl-L- 3- butylglycine Proline Chlorobenzaldoxime27 N—BOC-L-tert- Cyclobutaneacetic 3- butylglycine acidChlorobenzaldoxime 43 N—BOC-L-tert- 2-Cyclohexylacetic 2,4-Dimethoxy-5-butylglycine acid chlorobenzaldoxime 48 N—BOC-L-tert- 2-Norbornaneacetic2,4-Dimethoxy-5- butylglycine acid chlorobenzaldoxime 50 N—BOC-L-tert-2-Cyclohexylacetic 3- butylglycine acid Chlorobenzaldoxime 59N—BOC-L-tert- 2-cycloheptylacetic 2,4-Dimethoxy-5- butylglycine acidchlorobenzaldoxime 63 N—BOC-L-tert- 2-Cyclohexylacetic 3- butylglycineacid Chlorobenzaldoxime 67 N—BOC-L-tert- 2-(tetrahydro-2H-3-Chloro-5-fluoro-4- butylglycine pyran-4-yl)acetic ethoxybenzaldoximeacid 86 N—BOC-L-tert- Isopropyl isocyanate Piperonal oxime butylglycine90 N—BOC-L-tert- N/A 3- butylglycine Chlorobenzaldoxime 104N—BOC-L-tert- Tert-butylacetic acid 2,4-Dimethoxy-5- butylglycinechlorobenzaldoxime 105 N—BOC-L-tert- N/A 3- butylglycineChlorobenzaldoxime 117 N—BOC-L-tert- 4-methyltetrahydro- 3- butylglycine2H-pyran-4- Chlorobenzaldoxime carboxylic acid 121 N—BOC-L-tert- 2-(2,2-3- butylglycine dimethyltetrahydro- Chlorobenzaldoxime 2H-pyran-4-yl)acetic acid 126 N—BOC-L-tert- 2-Cyclohexylacetic 3-Chloro-5-fluoro-4-butylglycine acid ethoxybenzaldoxime 129 N—BOC-L-tert-2-cycloheptylacetic 2,4-Dimethoxy-5- butylglycine acidchlorobenzaldoxime 131 N—BOC-L-tert- N/A 2,4-Dimethoxy-5- butylglycinechlorobenzaldoxime 136 N—BOC-L-tert- 2-Cyclohexylacetic3-Chloro-5-fluoro-4- butylglycine acid ethoxybenzaldoxime 145N—BOC-L-tert- 2-(tetrahydro-2H- Piperonal oxime butylglycinepyran4-yl)acetic acid 153 N—BOC-L-tert- 2-((2R,5R)-2,5- 3- butylglycinedimethyltetrahydro- Chlorobenzaldoxime 2H-pyran-4- yl)acetic acid 168N—BOC-L-tert- 2-Cyclohexylacetic Thiophene-3- butylglycine acidcarboxaldehyde 172 N-Phenyl-L-tert- N/A 3- butylglycinechlorobenzaldoxime 178 N—BOC-L-tert- 2-(tetrahydro-2H-3-Chloro-5-fluoro-4- butylglycine pyran-4-yl)acetic ethoxybenzaldoximeacid 184 N—BOC-L-tert- 4-methyltetrahydro- 3- butylglycine 2H-pyran-4-Chlorobenzaldoxime carboxylic acid 188 N—BOC-L-tert- 2-Cyclohexylacetic3- butylglycine acid Chlorobenzaldoxime 192 N—BOC-L-tert- cyclopentyl2,5- 2,4-Dimethoxy-5- butylglycine dioxopyrrolidin-1-ylchlorobenzaldoxime carbonate 195 N—BOC-L-tert- 2-cyclohexylacetic3-chloro-4-methoxy- butylglycine acid 5- methylbenzaldoxime 2112-(tert-butoxy- 2-cyclohexylacetic 3- carbonylamino)- acidchlorobenzaldoxime 2-(1-methoxy- cyclopropyl)acetic acid 212N—BOC-L-tert- (S)-2-cyclohexyl-3- 3- butylglycine (1H-1,2,3-triazol-4-chlorobenzaldoxime yl)propanoic acid 214 N-(3- N/A 3- methoxyphenyl)-L-chlorobenzaldoxime tert-butylglycine 217 N—BOC-L-tert-2-Cyclohexylacetic 3- butylglycine acid Chlorobenzaldoxime 219N—BOC-L-tert- 2-cycloheptylacetic 3- butylglycine acidChlorobenzaldoxime 225 N—BOC-L-tert- cyclopentyl 2,5- 3- butylglycinedioxopyrrolidin-1-yl Chlorobenzaldoxime carbonate 231 N—BOC-L-tert-2-Cyclohexylacetic 3- butylglycine acid Chlorobenzaldoxime 233N—BOC-L-tert- 2-(1- 3- butylglycine hydroxycyclohexyl)Chlorobenzaldoxime acetic acid 247 N—BOC-L-tert- tert-Butyl isocyanate3- butylglycine Chlorobenzaldoxime 256 N—BOC-L-tert- 2-cyclohexylacetic5-Ethyl-2- butylglycine acid furaldoxime 263 N—BOC-L-tert-2-Cyclohexylacetic 3-Chloro-5-fluoro-4- butylglycine acidethoxybenzaldoxime 264 N—BOC-L-tert- N/A 2,4-Dimethoxy-5- butylglycinechlorobenzaldoxime 266 N—BOC-L-tert- (S)-2-cyclohexyl- 3- butylglycinepent-4-ynoic acid chlorobenzaldoxime 268 N—BOC-L-tert-2-Cyclohexylacetic 3- butylglycine acid Chlorobenzaldoxime 273N—BOC-L-tert- 2-Cyclohexylacetic 3- butylglycine acid Chlorobenzaldoxime280 (S)-2-isopropyl-4- N/A 3- (isopropylamino)- chlorobenzaldoxime4-oxobutanoic acid 282 N—BOC-L-tert- 2-(tetrahydro-2H- 2,4-Dimethoxy-5-butylglycine pyran-4-yl)acetic Chlorobenzaldoxime acid 284 N—BOC-L-tert-(S)-2- 3- butylglycine cyclohexylpropanoic chlorobenzaldoxime acid 286N—BOC-L-tert- 2-(4-methyltetra- 2,4-Dimethoxy-5- butylglycinehydro-2H-pyran-4- chlorobenzaldoxime yl)acetic acid 290 N—CBZ-L-tert-N/A Piperonal oxime butylglycine 294 N—BOC-L-tert- 2-cyclohexylacetic 3-butylglycine acid chlorobenzaldoxime 295 N-((S)- N/A Piperonal oximetetrahydrofuran- 3-yloxy)carbonyl)- L-tert-butylglycine 297N—BOC-L-tert- Tert-butylacetic acid 2,4-Dimethoxy-5- butylglycinechlorobenzaldoxime 307 N—BOC-L-tert- 2-Cyclohexylacetic 2,4-Dimethoxy-5-butylglycine acid chlorobenzaldoxime 310 N—BOC-L-tert- 2-(tetrahydro-2H-3,5-Dimethyl-4- butylglycine pyran-4-yl)acetic methoxy- acid benzalehyde326 N—BOC-L-tert- 2-(tetrahydro-2H- 2,4-Dimethoxy-5- butylglycinepyran-4-yl)acetic Chlorobenzaldoxime acid 335 N—CBZ-L-tert- N/A2,4-Dimethoxy- butylglycine benzaldoxime 337 N—BOC-L-tert- cyclopentyl2,5- 3- butylglycine dioxopyrrolidin-1-yl Chlorobenzaldoxime carbonate344 N—BOC-L-tert- N—FMOC-L- 3- butylglycine cyclohexylglycineChlorobenzaldoxime followed by 2- pyrazine carboxylic acid 346N—BOC-L-tert- 2-Norbornaneacetic 3- butylglycine acid Chlorobenzaldoxime351 N/A Tert-butylacetic acid 3- chlorobenzaldoxime 356 N—BOC-L-tert-2-(4-methyltetra- 2,4-Dimethoxy-5- butylglycine hydro-2H-pyran-4-chlorobenzaldoxime yl)acetic acid 362 N—BOC-L-tert- 2-Cyclohexylacetic3,5-Dimethyl-4- butylglycine acid methoxy- benzalehyde 369 N—BOC-L-tert-cyclopentyl 2,5- 3- butylglycine dioxopyrrolidin-1-yl Chlorobenzaldoximecarbonate 375 N—BOC-L-tert- 2-(tetrahydro-2H- Piperonal oximebutylglycine pyran-4-yl)acetic acid 382 N—BOC-L-tert-isopropylisocyanate Piperonal oxime butylglycine 388 N—BOC-L-tert-Cyclohexylacetic 3- butylglycine acid Chlorobenzaldoxime 411N—BOC-L-tert- 2-(tetrahydro-2H- 3-Chloro-5-fluoro-4- butylglycinepyran-4-yl)acetic ethoxybenzaldoxime acid 415 N—CBZ-L-tert- N/APiperonal oxime butylglycine 418 N—BOC-L-tert- 2-((2S,5R)-2,5- 3-butylglycine dimethyltetrahydro- Chlorobenzaldoxime 2H-pyran-4-yl)acetic acid 419 N—BOC-L-tert- 2-(2,2- 3- butylglycinedimethyltetrahydro- Chlorobenzaldoxime 2H-pyran-4- yl)acetic acid 440N—BOC-L-tert- cyclopentyl 2,5- 2,4-Dimethoxy-5- butylglycinedioxopyrrolidin-1-yl chlorobenzaldoxime carbonate 442 N—BOC-L-tert-2-((2S,5R)-2,5- 3- butylglycine dimethyltetrahydro- Chlorobenzaldoxime2H-pyran-4- yl)acetic acid 445 N—BOC-L-tert- 2-(1,4-dioxa- 3-butylglycine spiro[4.5]decan- Chlorobenzaldoxime 8-yl)acetic acid 446N—BOC-L-tert- 2-Norbornaneacetic 2,4-Dimethoxy-5- butylglycine acidchlorobenzaldoxime 453 N—BOC-L-tert- 2-Cyclohexylacetic 3- butylglycineacid Chlorobenzaldoxime 468 N—BOC-L-tert- 2-((2R,5R)-2,5- 3-butylglycine dimethyltetrahydro- Chlorobenzaldoxime 2H-pyran-4-yl)acetic acid 473 N—BOC-L-tert- Tert-butylacetic acid 2,4-Dimethoxy-5-butylglycine chlorobenzaldoxime 485 N—BOC-L-tert- trans-2-phenyl-1- 3-butylglycine cyclopropane- Chlorobenzaldoxime carboxylic acid 502N—BOC-L-tert- N—FMOC-L- 3- butylglycine cyclohexylglycineChlorobenzaldoxime followed by 2- pyrazine carboxylic acid 510N—BOC-L-tert- 2-(tetrahydro-2H- 2,4-Dimethoxy-5- butylglycinepyran-4-yl)acetic Chlorobenzaldoxime acid

Certain other compounds of Formula I may be prepared by Method 6 asillustrated below.

Method 6:

Referring to Method 6, the intermediate A1 is converted to theBoc-methyl ester F1. Removal of the Boc group from F1 provides theamine-ester F2 which is reacted with an R₁ carboxylic acid in thepresence of a coupling reagent to provide F3 wherein R₁ is R₄C(O)—. F3reacts with a nitrile oxide 1f to provide the spiroisoxazoline acid E4after hydrolysis of the corresponding methyl ester E3. Conversion of E4to E7 is achieved as described in Method 5a.

EXAMPLE 17 Compound No. 267

4-Hydroxy-3,5-dimethylbenzaldehyde (2.5 g, 16.6 mmol) in THF (100 mL)was treated with KOH (1.5 eq. of 1 N aq. solution, 25 mL) and2-iodopropane (2.0 eq.) and heated at reflux for 5 days. The reactionwas then cooled, transferred to a separatory funnel, diluted with MTBE,washed with H₂O, 1 N NaOH (twice), 0.5 N HCl (aq.), brine, dried overMgSO₄ and concentrated. The product was purified over silica gel on anISCO combiflash to yield 1.99 g (10.34 mmol)4-isopropoxy-3,5-dimethylbenzaldehyde as a colorless liquid. H¹ NMR (300MHz, CDCl3) 9.89 (s, 1H), 7.55 (s, 2H), 4.41-4.26 (m, 1H), 2.32 (s, 6H),1.32 (d, J=6 Hz, 6H).

4-(Isopropoxy)-3,5-dimethylbenzaldehyde (1.98 g, 10.3 mmol) in EtOH (60mL) was heated to 60° C. with hydroxylamine hydrochloride (2.4 M aq.solution, 5.2 mL, 1.2 eq.) and Na₂CO₃ (1.2 M solution, 5.2 mL, 0.6 eq.)at room temperature for 2 hours. The reaction was transferred to aseparatory funnel, diluted with EtOAc; the organic layer was separated,washed with brine, dried (MgSO₄), filtered and concentrated to yield 710mg (3.24 mmol) of 4-(isopropoxy)-3,5-dimethylbenzaldehyde oxime as alight yellow oil. ¹H-NMR (500 MHz, CDCl₃): 8.10 (s, 1H), 7.23 (s, 2H),4.29-4.18 (m, 1H), 2.29 (s, 6H), 1.29 (d, 6H).

4-(Isopropoxy)-3,5-dimethylbenzaldehyde oxime (166 mg, 0.801 mmol) inDMF (3 mL) at room temperature was stirred overnight with NCS (130 mg,0.974 mmol). To this reaction was added the methyl ester (257 mg, 0.679mmol) in DMF (1.5 mL) and triethylamine (1.2 eq.). This was stirredovernight at room temperature. The reaction was then diluted withEtOAc/Hexanes (4:1) and washed with 1N HCl (aq.). The layers wereseparated and the aqueous layer was back extracted with EtOAc/Hexanes(4:1). The organic layers were combined, washed with brine, dried(MgSO4), and concentrated. The compound was purified over silica gel onan ISCO Combiflash with EtOAc/Hexanes as eluent to yield 173 mg (0.296mmol) of compound 17A as a white solid. LCMS (M+1)=584.3

The compound 17A (173 mg, 0.30 mmol) was stirred with LiOH.H₂O (1.1 eq.)in THF/MeOH/H₂O (4:1:1, 3 mL) at RT overnight. The reaction was dilutedwith EtOAc, acidified with 1N HCl (aq) and the layers were separated.The aqueous layer was back extracted with EtOAc, the organic layerscombined, washed with brine, dried (MgSO₄) and concentrated to yield 171mg (0.30 mmol) of compound 17B as a white solid. FIA MS (M+1)=570.3.

Carboxylic acid 17B (83 mg, 0.146 mmol), EDC•HCl (37 mg, 1.3 eq.), HOBt(26 mg, 1.3 eq.), (3S)-3-amino-N-cyclopropyl-2-hydroxyhexanamidehydrochloride (64 mg, 2.0 eq.), and DIEA (0.100 mL, 4.0 eq.) werestirred in DMF (0.9 mL) at room temperature overnight. The reactionmixture was then diluted with EtOAc and washed with 1 N HCl (aq)(twice). The aqueous layer was separated and back extracted with EtOAc.The organic layers were combined, washed with brine, dried (MgSO₄), andconcentrated. The product was purified over silica gel on an ISCOcombiflash to yield 85 mg (0.115 mmol) of compound 17C. LCMS (M+1)=738.3

Compound 17C (85 mg, 0.115 mmol) in CH₂Cl₂ (1.0 mL) was treated withDess-Martin periodinane (54 mg, 1.1 eq.) for 30 minutes. The reactionwas quenched with equal volumes (˜1 mL) of saturated aqueous NaHCO₃ and1 N Na₂S₂O₃ (aq). The organic layer was separated and purified directlyover silica gel on an ISCO combiflash to yield 77 mg (0.105 mmol) ofCompound No. 267. FIA MS (M+1)=736.2. ¹H-NMR (300 MHz, CDCl₃): 7.33-7.26(m, 2H), 7.12 (d, 1H), 6.91 (d, 1H), 6.12 (d, 1H), 5.45-5.32 (m, 1H),4.78-4.63 (m, 2H), 4.29-4.17 (m, 2H), 3.71 (d, 1H), 3.43 (d, 1H), 3.30(d, 1H), 2.86-2.74 (m, 1H), 2.63-2.42 (m, 2H), 2.29 (s, 6H), 2.19-1.85(m, 3H), 1.84-0.82 (m, 34H), 0.65-0.58 (m, 2H).

EXAMPLE 18 Compound No. 556

4-Ethoxybenzaldehyde oxime (204 mg, 1.24 mmol), was dissolved in DMF (to0.2 M) and treated with NCS (1 eq.). The reaction was stirred untilstarting material was consumed. One half of the reaction volume wasremoved and treated with additional NCS (1.5 eq.) and stirred overnight.To this solution was then added the methyl ester (200 mg, 0.85 eq.) inDMF (0.3 mL) and triethylamine (0.10 mL, 1.1 eq.). The reaction wasstirred overnight at room temperature, then diluted with EtOAc, washedwith 1 N HCl (aq.), and washed with brine. The aqueous layer was backextracted with EtOAc and the combined organic layers were washed withbrine, dried (MgSO₄), and concentrated to a dark oil. The product waspurified over silica gel on an ISCO combiflash to yield 97 mg (0.168mmol) of compound 18A. LCMS (M+1)=576.3

Compound 18A (97 mg, 0.168 mmol) was dissolved in THF/MeOH/H2O (8:1:1, 5mL) and treated with LiOH.H2O (1.1 eq.) at room temperature overnight.The reaction was concentrated, diluted in EtOAc and methanol and washedwith 1N HCl (aq). The aqueous layer was separated and extracted withEtOAc. The combined organic layers were washed with brine, dried overMgSO₄ and concentrated to yield 76 mg (0.135 mmol) of compound 18B. FIAMS (M−1)=560.4

Compound 18B (35 mg, 0.062 mmol), EDC•HCl (15 mg, 1.3 eq.), HOBt (12 mg,1.3 eq.), an amino alcohol hydrochloride (55 mg, 2.0 eq.), and DIEA(0.044 mL, 4.0 eq.) were stirred in DMF (0.7 mL) at room temperatureovernight. The reaction was then diluted with EtOAc and washed with 1 NHCl (aq) (twice). The aqueous layer was separated and back extractedwith EtOAc. The organic layers were combined, washed with brine, dried(MgSO₄), and concentrated. The product was purified over silica gel onan ISCO combiflash to yield 28 mg (0.038 mmol) of compound 18C. LCMS(M+1)=730.2

Compound 18C (28 mg, 0.038 mmol) in CH₂Cl₂ (0.7 mL) was treated withDess-Martin periodinane (18 mg, 1.1 eq.) for 30 minutes. The reactionwas quenched with equal volumes (˜1 mL) of saturated aqueous NaHCO₃ and1N Na₂S₂O₃ (aq.). The organic layer was separated and purified directlyover silica gel on an ISCO Optix 10× to yield 24 mg (0.033 mmol) ofCompound No. 556. HA MS (M+1)=728.2. ¹H-NMR (300 MHz, CDCl₃): 7.65 (d,1H), 7.48 (dd, 1H), 7.11 (d, 1H), 6.95-6.88 (m, 2H), 6.08 (d, 1H),5.40-5.31 (m, 2H), 4.78-4.63 (m, 2H), 4.26 (d, 1H), 4.20-4.11 (m, 2H),3.71 (d, 1H), 3.42 (d, 1H), 3.27 (d, 1H), 2.84-2.73 (m, 1H), 2.63-2.46(m, 2H), 2.20-1.86 (m, 3H), 1.62-0.85 (m, 30H), 0.66-0.58 (m, 2H)

Listed below in Table 6 are additional compounds of Formula I preparedby Method 6.

TABLE 6 Additional Compounds of Formula I Prepared by Method 6. Com-pound Starting Starting Starting No. Material for P¹ Material for C¹Material for R₃ 18 N—BOC-L-tert- 2-Cyclohexyl- 7-Chloro-2,3-butylglycine acetic acid dihydrobenzo[b]furan-5- carboxaldoxime 19N—BOC-L-tert- 2-Cyclohexyl- 3-Chloro-4- butylglycine acetic acidmethylbenzaldoxime 28 N—BOC-L-tert- 2-Cyclohexyl- 2-Cyanobenzaldoximebutylglycine acetic acid 31 N—BOC-L-tert- 2-cyclohexyl-8-Quinoline-carbaldoxime butylglycine acetic acid 38 N—BOC-L-tert-2-Cyclohexyl- 2,5-Dichloro-3- butylglycine acetic acidmethoxybenzaldoxime 42 N—BOC-L-tert- 2-Cyclohexyl-8-Quinolinecarboxaldoxime butylglycine acetic acid 62 N—BOC-L-tert-2-Cyclohexyl- 5-chloro-3- butylglycine acetic acidThiophenecarboxaldoxime 68 N—BOC-L-tert- 2-cyclohexyl-8-Quinoline-carbaldoxime butylglycine acetic acid 74 N—BOC-L-tert-2-Cyclohexyl- 8-chloro-2,2- butylglycine acetic acid dimethylchromane-6-carbaldoxime 89 N—BOC-L-tert- 2-Cyclohexyl- 3-nitrobenzaldoximebutylglycine acetic acid 97 N—BOC-L-tert- 2-cyclohexyl- 3-Chloro-4-butylglycine acetic acid isopropoxybenzaldoxime 111 N—BOC-L-tert-2-Cyclohexyl- 3-Chloro-4-methoxy-5- butylglycine acetic acidmethylbenzaldoxime 114 N—BOC-L-tert- 2-Cyclohexyl- 3-Chloro-4-methoxy-5-butylglycine acetic acid methylbenzaldoxime 132 N—BOC-L-tert-2-Cyclohexyl- 5-Chloro-nicotinaldoxime butylglycine acetic acid 134N—BOC-L-tert- 2-Cyclohexyl- 5-Chloro-2,3- butylglycine acetic aciddihydrobenzo[b]furan-7- carboxaldoxime 158 N—BOC-L-tert- 2-Cyclohexyl-3-Chloro-6- butylglycine acetic acid methoxybenzaldoxime 165N—BOC-L-tert- 2-Cyclohexyl- 5-Chloro-2- butylglycine acetic acidmethoxynicotinaldoxime 168 N—BOC-L-tert- 2-Cyclohexyl- 3- butylglycineacetic acid Thiophenecarboxaldoxime 169 N—BOC-L-tert- 2-Cyclohexyl-3-Chloro-2- butylglycine acetic acid fluorobenzaldoxime 170N—BOC-L-tert- 2-Cyclohexyl- 5-Chloro-2,2-dimethyl-2,3- butylglycineacetic acid dihydrobenzo[b]furan-7- carboxaldoxime 250 N—BOC-L-tert-2-Cyclohexyl- 3-Chloro-5- butylglycine acetic acid methoxybenzaldoxime267 N—BOC-L-tert- 2-cyclohexyl- 4-Isopropoxy-3,5- butylglycine aceticacid dimethylbenzaldoxime 292 N—BOC-L-tert- 2-Cyclohexyl-5-Chloro-nicotinaldoxime butylglycine acetic acid 305 N—BOC-L-tert-2-Cyclohexyl- 6-Fluoro-1,3-benzodioxene- butylglycine acetic acid8-carbaldoxime 312 N—BOC-L-tert- 2-Cyclohexyl- 5-Chloro-6- butylglycineacetic acid methoxynicotinaldoxime 315 N—BOC-L-tert- 2-Cyclohexyl-5-Chloro-4-Methyl-3,4- butylglycine acetic acid dihydro-2H-1,4-benzoxazine-7- carbaldoxime 321 N—BOC-L-tert- 2-Cyclohexyl-5-Chloro-2,3-dimethoxy- butylglycine acetic acid benzaldoxime 366N—BOC-L-tert- 2-Cyclohexyl- 5-Chloro-4-methoxy-2- butylglycine aceticacid methylbenzaldoxime 370 N—BOC-L-tert- 2-Cyclohexyl-5-Chloro-piperonal oxime butylglycine acetic acid 396 N—BOC-L-tert-2-Cyclohexyl- 3-Chloro-5- butylglycine acetic acid methylbenzaldoxime406 N—BOC-L-tert- 2-cyclohexyl- 4-Cyclopropylmethoxy-3,5- butylglycineacetic acid dimethylbenzaldoxime 430 N—BOC-L-tert- 2-Cyclohexyl-8-Chloro-1-methyl-1,2,3,4- butylglycine acetic acidtetrahydro-quinoline-6- carbaldoxime 469 N—BOC-L-tert- 2-Cyclohexyl-2-Methoxy-nicotinaldoxime butylglycine acetic acid 478 N—BOC-L-tert-2-Cyclohexyl- 5-Chloro-2- butylglycine acetic acidthiophenecarboxaldoxime 494 N—BOC-L-tert- 2-Cyclohexyl- 3-Chloro-4,5-butylglycine acetic acid dimethoxybenzaldoxime 499 N—BOC-L-tert-2-Cyclohexyl- 7-Chloro-2,3- butylglycine acetic aciddihydrobenzo[b]furan-5- carboxaldoxime 500 N—BOC-L-tert- 2-Cyclohexyl-4-Methoxy-3- butylglycine acetic acid methylbenzaldoxime 513N—BOC-L-tert- 2-cyclohexyl- 4-Ethoxy-3,5- butylglycine acetic aciddimethylbenzaldoxime 556 N—BOC-L-tert- 2-cyclohexyl- 3-Chloro-4-butylglycine acetic acid ethoxybenzaldoxime 591 N—BOC-L-tert-2-Cyclohexyl- 2-Pyridinecarboxaldoxime butylglycine acetic acid 592N—BOC-L-tert- 2-Cyclohexyl- 2-Pyridinecarboxaldoxime butylglycine aceticacid 593 N—BOC-L-tert- 2-Cyclohexyl- 4-Chloro-2- butylglycine aceticacid pyridinecarboxaldoxime 594 N—BOC-L-tert- 2-Cyclohexyl- 3-Chloro-6-butylglycine acetic acid fluorobenzaldoxime

Certain other compounds of the invention may be prepared by Method 7 asillustrated below.

Method 7:

Referring to Method 7, the Cbz hydroxy acid G1 is converted to themethyl ester G2 and deprotected to provide the amino-ester G3. Reactionof G3 with the spiroisoxazoline acid G4 in the presence of a couplingreagent provides the intermediate G5. Hydrolysis of the methyl ester ofG5 provides the hydroxy acid G6 which is oxidized with, for example,Dess-Martin periodinane to provide the ketoacid G7. Reaction of G7 withan amine R₁₃R₁₀NH in the presence of a coupling reagent provides thefinal product G8.

EXAMPLE 19 Compound No. 275

Step 1: Preparation of Compound Q.

1.00 g of acid 19A was dissolved in 14 mL of methanol and heated toreflux. Two drops of concentrated H₂SO₄ was added and the reactionrefluxed overnight. The mixture was cooled to room temperature, andneutralized with 50 mL of NaHCO₃ (sat. aq.). The reaction mixture wasextracted three times with 50 mL of ethyl acetate. The combined organicextracts were dried over magnesium sulfate and evaporated to yield 1.01g of compound 19B as a white powder. Major diastereomer ¹H-NMR (300 MHz,CDCl₃) δ: 7.40-7.31 (m, 5H), 5.12 (s, 2H), 4.99 (d, 1H, J=8.7 Hz), 4.35(s, 1H), 4.15-4.02 (m, 1), 3.81 (s, 3H), 3.05 (br s, 1H), 1.67-1.17 (m,4H), 0.91 (t, 3H, J=6.8 Hz). Minor diastereomer ¹H-NMR (300 MHz, CDCl3)δ: 7.40-7.31 (m, 5H), 5.07 (s, 2H), 4.90 (d, 1H, J=9.8 Hz), 4.19 (s,1H), 4.15-4.02 (m, 1H), 3.76 (s, 3H), 3.03 (br s, 1H), 1.67-1.17 (m,4H), 0.96 (t, 3H, J=7.1 Hz).

1.00 g of CBz-protected methyl ester 19B was dissolved in 11 mL ofmethanol. 150 mg of Pd(OH)₂ (20 wt % on carbon) was added, and themixture flushed with 1 atm of hydrogen gas and stirred at roomtemperature for 3 hours. The methanolic solution was filtered through aCelite® plug and the filter pad rinsed with additional methanol. Uponevaporation, a light yellow oil was collected and redissolved in 5 mL ofDCM and treated with 1.5 mL of 4 M HCl solution in dioxane. Uponstiffing for 1 minute, the reaction was evaporated. 0.65 g of compound19C was collected as a white powder, and characterized by LCMS(M+1=162.0).

0.80 g of the spiroisoxazoline acid of compound 19D was stirred with0.33 g of HOBt, 0.81 g of HBTU, and 15 mL of DMF. To the stirringsolution was added 807 μL of DIPEA, and stirred for 10 minutes. 0.33 gof the hydrochloride salt 19C was added. The reaction was stirred atroom temperature for 3 hours. To the reaction mixture was added 200 mLof EtOAc, and the mixture washed twice with 100 mL of NaHCO₃ (sat. aq.),then 100 mL of brine. The organic phase was dried over MgSO₄ andevaporated. The crude reaction mixture was purified by elution throughsilica gel column (40 g column, gradient elution, 40-55% EtOAc:Hexanes)to give 1.02 g of compound 19E as a white powder, which was identifiedby LCMS (M+1=661.3).

1.04 g of methyl ester 19E was stirred in 6 mL of THF and to thissolution was added 3 mL of 1 M LiOH(aq). The reaction was stirred atroom temperature for 2 hours where it was determined by HPLC to becomplete. The reaction was treated with 6 mL of 1 M HCl, and extractedthree times with 15 mL of ethyl acetate. The combined extracts wereevaporated to give 1.00 g of compound Q as a beige solid which wascarried on to the next step.

Step 2: Preparation of Compound R

To a solution of compound Q (0.300 g, 0.46 mmol) in CH₂Cl₂ (15 mL) wasadded 5.58 mL of a 0.16 M solution of Dess Martin periodinane in CH₂Cl₂dropwise. After it was stirred for 4 hours at room temperature, 10 mL of1M Na₂S₂O₃ solution was added and the reaction mixture was stirred for30 minutes at ambient temperature. The organic layer was separated,washed with water, dried over Na₂SO₄, filtered and concentrated. Thecrude mixture was redissolved in CH₂Cl₂ and precipitated with Hexanesand filtered to give 230 mg of compound R. LC/MS: m/z 645.7 (M+H)⁺ at1.99 minutes (10-99% CH₃CN (0.035% TFA)/H₂O (0.05% TFA))

Step 3: Preparation of Compound No. 275

To a suspension of compound R (20 mg, 0.0.031 mmol) in anhydrousacetonitrile was added pyridine (10 μL, 0.124 mmol),2-chloro-l-methyl-pyridinium iodide (15.3 mg, 0.06 mmol), HOBt (6.8 mg,0.05 mmol), followed by the addition of a 50 μL solution ofisopropylamine (3.7 mg, 0.062 mmol) in anhydrous acetonitrile. Thereaction was allowed to stir at room temperature and complete after twohours. The reaction mixture was quenched with 1 mL of saturated aqueoussodium bicarbonate solution, the layers were separated and aqueous layerwas extracted three times with CH₂Cl₂. The combined organics were driedover Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was dissolved in 1.5 mL CH₂Cl₂ and purified by normal phase HPLC(10-99% EtOAc/Hexanes) to yield Compound No. 275. LC/MS: m/z 686.7(M+H)⁺ at 2.01 minutes (10-99% CH₃CN (0.035% TFA)/H₂O (0.05% TFA))

EXAMPLE 20 Compound No. 181

To a suspension of R (20 mg, 0.031 mmol) in anhydrous 1,4-dioxane wasadded pyridine (7.6 μL, 0.093 mmol), then pentafluorophenyltrifluoroacetate (8.8 μL, 0.05 mmol) and allowed to stir for 1.5 hoursat room temperature, upon which 7-amino-4-methyl-1H-quinolin-2-one (14mg, 0.08 mmol) was added. The reaction was allowed to stir at roomtemperature and complete after one hour. The reaction mixture wasquenched with 1 mL of saturated aqueous sodium bicarbonate solution, thelayers were separated and aqueous layer was extracted three times withCH₂Cl₂. The combined organics were dried over Na2SO4, filtered andconcentrated under reduced pressure. The residue was dissolved in 1.5 mLCH₂Cl₂ and purified by normal phase HPLC (10-99% EtOAc/Hexanes) to yieldCompound No. 181. LC/MS: m/z 801.7 (M+H)⁺ at 2.06 minutes (10-99% CH₃CN(0.035% TFA)/H₂O (0.05% TFA)).

EXAMPLE 21 Compound No. 605

A mixture of(3S)-3-((5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-1-oxa-2,7-diazaspiro[4.4]non-2-enecarboxamido)-2-hydroxyhexanoicacid (0.02 g, 0.03 mmol), (3,5-dimethoxyphenyl)methanamine (5.68 mg,0.033 mmol), HOBt (6.8 mg, 0.05 mmol), DIPEA (22 μL, 0.124 mmol) andCH₂Cl₂ (70 μL) was stirred at room temperature for 10 minutes. To themixture was then added a solution of Mukaiyama's reagent(2-chloro-1-[4-(1H,1H,2H,2H-perfluoro-9-methyldecyl)benzyl]pyridiniumhexafluorophosphate) in 200 μL of acetonitrile and the reaction wasstirred at room temperature. After 5 hours, 1.34 mL of 0.3 M Dess-MartinPeriodinane in CH₂Cl₂ was added and the mixture stirred. After 2 hours,the oxidant was quenched with 1.0 mL of saturated NaHCO₃, 1 mL of 1NNa₂S₂O₃ and stirred vigorously. The organic layer was separated, driedover Na₂SO₄, filtered and concentrated. The residue was dissolved in 1.5mL CH₂Cl₂ and purified by normal phase HPLC (10%-99% Ethylacetate/Hexanes) to yield Compound No. 605,(5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((S)-1-(3,5-dimethoxybenzylamino)-1,2-dioxohexan-3-yl)-1-oxa-2,7-diazaspiro[4.4]non-2-ene-8-carboxamide.LC/MS: m/z 794.7 (M+H)⁺ at 4.11 minutes (10%-99% CH₃CN (0.035% TFA)/H₂O(0.05% TFA)).

Listed below in Table 7 are reagents used to prepare additionalcompounds of Formula I by Method 7.

TABLE 7 Reagents Used to Prepare Additional Compounds of Formula I byMethod 7. Compound No. R_(2z)R_(2w)NH 2 tert-butylamine 6 2-aminoindane17 benzo[d]thiazol-2-amine 49 3-((tetrahydrofuran-3-yl)methoxy)azetidine58 (R)-(+)-1-(3-methoxyphenyl)ethylamine 69 6-Methoxytryptamine 734-1H-pyrazol-1-yl-benzylamine 77 benzylamine 79 azetidine 842,5-dimethoxyaniline 91 (4-(4-methoxyphenyl)tetrahydro-2H-pyran-4-yl)methanamine 96 3-cyano-4-methylaniline 99 cyclohexylamine113 N,N-Diethylamine 120 Phenyl-2-pyridinemethylamine 1273′,5′-dimethoxybenzylamine 133 3-Ethoxyazetidine 1381-(3-(2-aminopropyl)-1H-indol-5-yl)ethanone 140 Ethylamine 1412,3-dihydro-1,4-benzodioxin-2-ylmethylamine 143 Isobutylamine 148N-(3-aminophenyl)methanesulfonamide 176(2-Phenyl-1,3-thiazol-4-yl)metylamine 1817-amino-4-methylquinolin-2(1H)-one 182 N-Methylethylamine 186(3R)-(+)-3-acetamidopyrrolidine 206beta-alanine-4-methoxy-betanaphthylamide 221N-ethyl-3,4-methylenedioxyamphetamine 238(R)-3-((tetrahydrofuran-2-yl)methoxy)azetidine 253 Dimethylamine 255(S)-(−)-1-(3-methoxyphenyl)ethylamine 265 cyclopropylmethylamine 275Isopropylamine 277 (S)-(+)-tetrahydrofurfurylamine 293 3-aminoisoxazole296 (S)-alpha-methylbenzylamine 298 3-Pyrazol-1-yl-benzylamine 3001-(Ethyl)propylamine 302 5-Methoxytryptamine 347(R)-(−)-2-(methoxymethyl)pyrrolidine 350 N-Methyl-N-propylamine 3553-Aminobenzamide 368 3-(tetrahydrofuran-3-yloxy)azetidine 372Cyclopentylamine 399 1-Aminocyclopropane-1-carboxylic acid methyl ester401 Cyclobutylamine 404 2-Methoxyethylamine 408 3-(Aminoethyl)pyridine410 Morpholine 426 3-Hydroxy-3-methylazetidine 4291-Phenylcyclopropylamine 433[3-(4-chlorophenyl0-5-isoxazolyl}methanamine 441 Furfurylamine 4472-(3-Pyridyl)ethylamine 452 (R)-2-Butylamine 4583-(2-aminoethyl)indolin-2-one 461 4-(Aminomethyl)pyridine 4792-Fluoroethylamine 488 2-methoxyphenoxyethylamine 493 Methylamine 496Pyrrolidine 507 (S)-2-Amino-1,1-dihenyl-1-propanol 508(S)-(+)-2-(methoxymethyl)pyrrolidine 521 3,3-difluoro-azetidine 537Propylamine 540 2-(3-methoxyphenyl)ethylamine 546(R)-alpha-methylbenzylamine 565

567 2-aminomethyl benzimidazole 568 Pipecoline 573 3,4-Difluoroaniline588 3-cyanoanilinePreparation of Non-Commercial Azetidines Listed in Table 7

N-Benzyhydryl-3-methanesulfonylazetidine (104 mg) was combined withethanol (1.0 mL) and heated in a sealed vial at 95° C. overnight. Thereaction was monitored by TLC (30% EtOAc:Hexane). Workup was conductedby adding 1 mL of saturated potassium carbonate solution, and extractingtwice with 0.5 mL of ethyl acetate. The combined organic extracts werepurified on silica (4 g column, gradient elution, 0-30% EtOAc:hexane).Yielded 49 mg of N-benzhydryl-3-ethoxyazetidine as a clear colorlessoil. LCMS (M+1=268.2).

N-Benzhydryl-3-ethoxyazetidine (49 mg) was dissolved in 1 mL ofmethanol. 22 mg of 10% Pd/C (Degussa-type) was added, and the reactionwas carried out under a hydrogen atmosphere. The reaction was stirred atroom temperature for 64 h. The mixture was filtered through the Celite®,washed thoroughly with methanol, and evaporated to give a yellow oil (30mg). The oil consists of a mixture of diphenylmethane and the freeazetidine. The crude oil mixture was carried onto subsequenttransformations and used in excess.

The following azetidines were prepared in a similar fashion as above, byusing the corresponding alcohols.

The azetidine

was prepared in the method described by Frigola, J. et al. in J. Med.Chem., 36 (1993), 801-810.

Certain other compounds of Formula I may be prepared by Method 8 asillustrated below.

Method 8:

Referring to Method 8, the spiroisoxazoline acid E4 reacts with theamino ester H1 in the presence of a coupling reagent to provide theintermediate H2. Macrocyclization of H2 results in compound H3.Hydrolysis of the ester H2 provides acid H4. Reaction of acid H4 with asulfonamide or sulfamide in the presence of a coupling reagent providesthe product H5.

EXAMPLE 22 Compound No. 409

(S)-2-(tert-butoxycarbonylamino)non-8-enoic acid, purchased from RSpAmino Acid located in Massachusetts, (179 mg, 1.0 eq.) was stirred inDMF with HBTU (376 mg, 1.5 eq.), HOBt (94 mg, 1.05 eq.), and DIEA (345uL, 3.0 eq.) for 15 minutes. Added compound 22A (194 mg, 1.0 eq.) andstirred overnight. To the solution was added ethyl acetate. The solutionwas washed with 1 N HCl (thrice) followed by brine, dried over sodiumsulfate, filtered, concentrated and purified by silica chromatography(10-30% ethyl acetate/hexanes gradient) to yield compound 22B (253 mg,70%). (M+H=548.2).

Compound 22B (253 mg, 1.0 eq.) was stirred in THF (1 mL) and methanol(0.5 mL). To the solution was added lithium hydroxide (97 mg, 5.0 eq.)in water (0.5 mL) and stirred for 2 more hours. The mixture was dilutedwith ethyl acetate, washed with 1 N HCl, then brine, and the solutionwas dried over MgSO₄, filtered and concentrated to yield compound 22C(235mg, 95%) as a pure white solid (M+H=534.2).

Compound 22C (247 mg, 1.0 eq.) stirred in 1 mL acetonitrile. To thesolution was added TBTU (297 mg, 2.0 eq.), DIEA (241 uL, 3.0 eq.), then(1R,2S)-methyl-1-amino-2-vinylcyclopropanecarboxylate (86 mg, 1.2 eq.)and stirred overnight. The solution was diluted with ethyl acetate andwashed with 1 N HCl then brine, dried over sodium sulfate, filtered,concentrated and purified by silica chromatography (10-70% ethylacetate/hexanes gradient) to yield compound 22D (230 mg, 76%).(M+H=657.2).

Compound 22D (230mg, 1.0eq.) was stirred in 70mL CH₂Cl₂ withHoveyda-Grubbs catalyst (22 mg, 0.1 eq.) at reflux for 1 hour, and thesolution cooled to room temperature and purified by silicachromatography (10-70% ethyl acetate/hexanes) to yield compound 22E (172mg, 77%)

Compound 22E (172 mg, 1.0 eq.) was stirred in THF (1 mL) and methanol(0.5 mL). To the solution was added LiOH (46 mg, 4.0 eq.) in 0.5 mLwater and solution stirred for 2 more hours. To the solution again wasadded ethyl acetate and washed with 1N HCl and brine, dried overmagnesium sulfate, filtered, and concentrated to yield compound 22F (155mg, 92%) as a pure white solid (M+H=617.1).

Compound 22F (155 mg, 1.0 eq.) stirred in 1 mL DMF withcarbonyldiimidazole (49 mg, 1.2 eq.) at 80° C. for 15 minutes. To thesolution was added cyclopropanesulfonamide (49 mg, 1.6 eq.) followed byDBU (36 uL, 1.0eq.) and stirred for another 10 minutes at 80° C. Then tothe solution was added ethyl acetate and solution washed with 1 N HCland brine, dried over MgSO₄, filtered, and concentrated. The product waspurified by silica chromatography (100% DCM to 5% methanol/DCM gradient)to give Compound No. 409 (64 mg, 35%). (M+H=718.1.)

Listed below in Table 8 are additional compounds of Formula I preparedby Method

TABLE 8 Additional Compounds of Formula I Prepared by Method 8 CompoundStarting No. Material for W Starting Material for R3 1 OH7-Chloro-2,3-dihydrobenzo[b]furan-5- carboxaldoxime 137 OH3-Chlorobenzaldoxime 163 Cyclopropane Phenylglyoxylohydroxamyl chloridesulfonamide 232 Cyclopropane 3-Chlorobenzaldoxime sulfonamide 320 OHPhenylglyoxylohydroxamyl chloride 386 Cyclopropane7-Chloro-2,3-dihydrobenzo[b]furan-5- sulfonamide carboxaldoxime 409Cyclopropane 3-Chlorobenzaldoxime sulfonamide 470 OH3-Chlorobenzaldoxime

Certain other compounds of Formula I may be prepared in Method 9 asillustrated below.

Method 9:

Referring to Method 9, the protected spiroisoxazoline B3 (prepared byMethod 2) reacts with the resin bound imino-amine D1 to provide theintermediate I1. Deprotection of I1 provides the amine I2 which reactswith an R₁ carboxylic acid in the presence of a coupling reagent toprovide I3 wherein R₁ is R₄C(O)—. Hydrolysis of I3 provides the finalcompound A10.

A person skilled in the art can use the examples and methods describedherein, along with known synthetic methodologies, to synthesizecompounds of Formula I according to Method 9 illustrated above.

Listed below in Table CC are additional compounds of Formula I preparedby Method 9.

TABLE CC Additional Compounds of Formula I Prepared by Method 9 CompoundNo. P¹ 46 5-Bromoindole-2-carboxylic acid 54 Acetyl-D-ethionine 602-(R)-[[(4-Methylphenyl)Sulfonyl]amino]- 2-phenylacetic acid 652-oxo-1-phenylpyrrolidine-3-carboxylic acid 88Acetyl-D-Methyltyrosine-OH 98 N-Acetyl-L-leucine 1002-[[(4-Fluorophenyl)Sulfonyl]amino]-3- methylbutanoic acid 1575,6-dimethoxyindole-2-carboxylic acid 218 Pyr-Val-OH 2271-carbamoylcyclopropanecarboxylic acid 2465-(2,4-dimethylphenylamino)-5- oxopentanoic acid 2484-Chloro-2-(6-methoxypyridin-3- ylamino)benzoic acid 3093-[[(4-acetamidophenyl)sulfonyl]amino]-3- propanoic acid 3283-(3,4-dihydroisoquinolin-2(1H)- ylsulfonyl)benzoic acid 332(S)-2-acetamido-3-(4- isopropoxyphenyl)propanoic acid 3763-(2-oxobenzo[d]oxazol-3(2H)-yl)propanoic acid 3804-trifluoromethoxyphenylacetic acid 3952-[[(4-Methoxyphenyl)Sulfonyl]amino]-3- methylbutanoic acid 3972-((S)-2-oxo-4-phenyloxazolidin-3-yl)acetic acid 412Acetyl-D-tyrosine-OH 416 2-(R)-[[(4-Chlorophenyl)Sulfonyl]amino]-3-methylpentanoic acid 420 3-(2-diethylamino)-2-oxoethyl)1H-indole-2-carboxylic acidi 421 trans-2-Phenyl-1-cyclopropanecarboxylic acid 4662-[[(4-Fluorophenyl)Sulfony]amino]-2- phenylacetic acid 4762-(S)-[[(4-Methylphenyl)Sulfonyl]amino]- 2-phenylacetic acid 4833-(N-Phenylphenylsulfonamido)propanoic acid 489 2-(R)-[[(4-Methoxyphenyl)Sulfonyl]amino]-3- methylbutanoic acid 5012-[(PhenylSulfonyl)amino]-2-phenylacetic acid 534 2-(R)-[[(4-Methoxyphenyl)Sulfonyl]amino]-3- methylpentanoic acid 5742-(1-oxoisoindolin-2-yl)propanoic acid 5866-(2,5-dimethoxyphenyl)-2-oxo-1,2,3,6- tetrahydropyrimidine-4-carboxylicacid 587 2-(R)-[[(4- Methoxyphenyl)Sulfonyl]amino]-4- methylpentanoicacid

Certain other compounds of Formua I may be prepared in Method 10 asillustrated below.

Method 10:

Referring to Method 10, the protected spiroisoxazoline B3 (e.g., R₁ isFmoc) reacts with M10A (R″, e.g., can be methyl or immobilized onPS-Wang resin) to provide intermediate M10B. Hydrolysis of M10B yieldsthe carboxylic acid M10C, which is subsequently coupled with theappropriate sulfonamide to afford the final compound M10D. M10C can alsobe a final compound of formula I.

Similarly, a person skilled in the art can use the examples and methodsdescribed herein, along with known synthetic methodologies, tosynthesize compounds of Formula I according to Method 10 illustratedabove.

Listed below in Table DD are additional compounds of Formula I preparedby Method 10.

TABLE DD Additional Compounds of Formula I Prepared by Method 10Compound P1 Starting C1 Starting No. W Starting Material MaterialMaterial R3 Starting Material 35 Cyclopropane N—Boc-L-tert- NA 3-sulfonamide butylglycine Chlorobenzaldoxime 45 Cyclopropane N—((S)- NA3- sulfonamide tetrahydrofuran- Chlorobenzaldoxime 3-yloxy)carbonyl)-L-tert-butylglycine 57 Cyclopropane N—Boc-L-tert- NA 7-Chloro-2,3-sulfonamide butylglycine dihydrobenzo[b]furan- 5-carboxaldoxime 115

N—Boc-L-tert- butylglycine 2- cyclohexylacetic acid 3-Chlorobenzaldoxime 130 Cyclopropanesulfonamide N-Alloc-L-tert- NA 3-butylglycine Chlorobenzaldoxime 144 OH N—Boc-L-tert- 2-cyclohexylacetic3- butylglycine acid Chlorobenzaldoxime 162 Cyclopropane N—Boc-L-tert-2-cyclohexylacetic 3- sulfonamide butylglycine acid Chlorobenzaldoxime190 Cyclopropane N—Boc-L-tert- cyclopentyl 2,5- 3- sulfonamidebutylglycine dioxopyrrolidin- Chlorobenzaldoxime 1-yl carbonate 269 OHN—((S)- NA 3- tetrahydrofuran- Chlorobenzaldoxime 3-yloxy) carbonyl)-L-tert-butylglycine 272 OH N—Boc-L-tert- cyclopentyl 2,5- Nitropropanebutylglycine dioxopyrrolidin- 1-yl carbonate 359 CyclopropaneN—Boc-L-tert- 2-(tetrahydro- 3- sulfonamide butylglycine 2H-pyran-4-Chlorobenzaldoxime yl)acetic acid 384 OH N—Boc-L-tert- 2-(tetrahydro- 3-butylglycine 2H-pyran-4- Chlorobenzaldoxime yl)acetic acid 438Cyclopropane N—Boc-L-tert- cyclopentyl 2,5- Nitropropane sulfonamidebutylglycine dioxopyrrolidin- 1-yl carbonate 439 OH N—Boc-L-tert- NA 3-butylglycine Chlorobenzaldoxime 443 OH N—Boc-L-tert- cyclopentyl 2,5- 3-butylglycine dioxopyrrolidin- Chlorobenzaldoxime 1-yl carbonate 457 OHN—Boc-L-tert- tert- 3- butylglycine Butylisocyanate Chlorobenzaldoxime460 OH N-Alloc-L-tert- NA 3- butylglycine Chlorobenzaldoxime

ADDITIONAL EXAMPLES EXAMPLE 23 Compound No. 610

Oxime 23A (6.29 g, 40 4 mmol) was dissolved in DMF (63 mL) andN-chlorosuccinimide (5.39 g, 40.4 mmol) was added portionwise to thestiffing solution. Stirring continued for 3 hours at room temperaturewhen conversion was determined to be 56% (by HPLC). The reaction waspushed to completion by gentle heating at 70° C. for 45 minutes.4-Methyleneproline derivative (8.81 g, 31.1 mmol) was added and rinsedinto the solution using DMF (5 mL). Triethylamine (5.7 mL) was carefullyadded dropwise over 30 minutes. The reaction was then stirred at roomtemperature for 16 hours overnight. An aliquot was analyzed by HPLC andit was determined to contain a 4:1 ratio of cycloaddition diasteromers.Ethyl acetate (200 mL) was added and the organic phase was washed withwater (thrice, 200 mL each) and brine (200 mL). The organic phase wasthen dried over magnesium sulfate and evaporated. The crude oil wasdivided into two portions and each was purified using an ISCO combiflashequipped with a 330 g silica column (10-20% EtOAc: pet. ether, 72minutes). The desired product was the major isomer which eluted from thecolumn ahead of the minor isomer and 9.42 g of 23B was obtained as anorange oil (69%). The minor isomer was also isolated, subjected to arecrystallization from EtOAc:hexane, and obtained as an off-whitecrystalline powder (1.53 g, 12%).

Compound 23B (9.42 g) was stirred in trifluoroacetic acid (12 mL) for 2hours. The solvent was evaporated and replaced with methanol (50 mL).The solution was heated to reflux and H₂SO₄ (3.0 mL) was added dropwise.The reaction was refluxed for a total of 6 hours when by HPLC,conversion to the methylester was determined to be greater than 95%. Thereaction was cooled and evaporated to remove the excess methanol. Theresulting oil was redissolved in CH₂Cl₂ (200 mL) and neutralized withsaturated sodium bicarbonate (200 mL). The organic phase was collected,and the aqueous phase was extracted with CH₂Cl₂ (twice, 100 mL each).The organic extracts were combined, evaporated over magnesium sulfate,and evaporated to give 5.09 g of compound 23C as an oil (80%) that wasimmediately carried onto the next step.

The amino ester 23C (1.25 g, 4.24 mmol) was treated with LiOH.H₂O (186mg, 4.4 mmol) in THF/H₂O (3:1, 10 mL) for 45 minutes. The solvents wereremoved in vacuo to obtain a solid. This solid was slurried in acetone(20 mL) and saturated NaHCO₃ (aq) (20 mL) at room temperature. Fmoc-Cl(1.12 g, 4.33 mmol) was added and the reation was monitored by HPLC.After 20 minutes, the contents of the reaction flask were transferred toa separatory funnel with CH₂Cl₂ and acidified with 2 N HCl (aq.). Theaqueous phase was extracted with CH₂Cl₂ (twice, 100 mL each). Theresulting emulsion was filtered, and the organic layers were combined,dried over MgSO₄, and concentrated to give compound 23D.

Compound XX4 was shaken in a solution of 20% piperidine in DMF (20 mL)for 60 minutes. The resin was washed with DMF (thrice), CH₂Cl₂ (thrice)and repeated. The resulting resin was then shaken with compound 23D (437mg, 0.87 mmol), HATU (392 mg, 1.03 mmol), and DIEA (0.300 mL, 1.72 mmol)in DMF (10 mL) overnight. The result compound bound resin 23F was thenwashed with DMF (thrice), CH₂Cl₂ (thrice) and repeated. (M+1)=612.26.

The combound bound resin 23F was shaken in 20% piperidine in DMF (8 mL)for 2 hours. The resin was then washed with DMF (thrice), CH₂Cl₂(thrice) and repeated. (M+1)=390.1. This resin was then shaken overnightin DMF with (S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoicacid (3 eq.), HOBT (3 eq.), HBTU (3 eq.), and DIEA (6 eq.). The resinwas washed with DMF (thrice) and CH₂Cl₂ (thrice) and repeated, thenshaken for 100 minutes in TFA (5 mL). The resulting resin was filteredand the filtrate concentrated and purified by reverse phasechromatography to yield 9.4 mg of compound Compound 443 as a whitesolid. (M+1)=615.6, ¹H-NMR (500 MHz, DMSO-d₆): 8.63 (s, 1H), 7.67 (s,1H), 7.63 (d, J=6.7 Hz, 1H), 7.55-7.49 (m, 2H), 6.90 (d, J=8.4 Hz, 1H),5.77-5.69 (m, 1H), 5.20-5.17 (m, 1H), 5.06 (d, J=10.5 Hz, 1H), 4.93(brs, 1H), 4.35 (t, J=7.7 Hz, 1H), 4.11 (d, J=8.8 Hz, 1H), 4.06 (d,J=10.9 Hz, 1H), 3.80 (d, J=11.6 Hz, 1H), 3.62-3.50 (m, 2H), 2.63-2.31(m, 2H), 2.18-2.13 (m, 1H), 2.07-2.01 (m, 1H), 1.87-1.51 (m, 9H),1.29-1.28 (m, 1H), 0.95-0.91 (brs, 9H).

Compound 23G (6.6 mg, 0.011mmol) was stirred in DMF (0.5 mL) with CDI(2.8 mg, 0.017 mmol) for 1 hour at 80° C. Cyclopropyl sulfonamide (3.8mg, 0.031 mmol) and DBU (0.01 mL) were added, the heat was removed andthe reaction was stirred overnight at room temperature. The reaction waspurified by reverse phase chromatography to yield 2.8 mg of Compound No.190 (0.0039 mmol). (M+1)=718.1. ¹H-NMR (500 MHz, methanol-d₄): 9.26 (s,0.4H), 9.02 (s, 0.6H), 7.72 (d, J=1.7 Hz, 1H), 7.61 (dd, J=1.3, 7.3 Hz,1H), 7.47-7.41 (m, 2H), 5.81 - 5.73 (m, 1H), 5.33-5.30 (m, 1H),5.14-5.10 (m, 1H), 5.03 (brs, 1H), 4.45-4.41 (m, 1H), 4.31-4.25 (m, 2H),3.94 (d, J=11.0 Hz, 1H), 3.62-3.53 (m, 2H), 2.99-2.92 (m, 1H), 2.55-2.49(m, 1H), 2.29-2.23 (m, 2H), 1.89-1.53 (m, 10H), 1.44-1.40 (m, 1H),1.32-1.24 (m, 1H), 1.19-1.02 (m, 2H), 0.90 (s, 9H).

EXAMPLE 24 Compound No. 618

Carboxylic acid 24A (69 mg, 0.13 mmol), HATU (50 mg, 0.13 mmol),compound 24B (0.13 mmol), and DIEA (0.045 mL, 0.26 mmol) were stirred inacetonitrile (1.5 mL) for 2 hours. The reaction was then diluted inEtOAc, washed with saturated NaHCO₃ (aq), brine, dried (MgSO4), andconcentrated. Purification on silica gel yielded 76 mg (0.12 mmol, 91%)of compound 24C. LCMS (M+1)=614.4.

The methyl ester 24C (76 mg, 0.12 mmol) dissolved in THF/H₂O (5:1, 2 mL)and stirred overnight with LiOH.H₂O (1.5 eq.). Acidified reaction with1N HCl (aq) and concentrated. Residue was dissolved in CH₂Cl₂/MeOH(93:7) and eluted through a plug of silica gel to yield 75 mg (0.11mmol) of Compound No. 144. LCMS (M+1=627.4). ¹H-NMR (500 MHz,Methanol-d₄): 7.84 (d, J=9.1 Hz, 0.5H), 7.71 (s, 1H), 7.60 (d, J=7.2 Hz,1H), 7.45-7.40 (m, 2H), 5.90-5.83 (m, 1H), 5.23 (d, J=1.4 Hz, 1H), 5.07(d, J=10.3 Hz, 1H), 4.60 (m, 1H), 4.52-4.49 (m, 1H), 4.27 (m, 1H), 3.90(m, 1H), 3.59-3.48 (m, 2H), 2.58 (dd, J=8.0, 12.6 Hz, 1H), 2.37-2.32 (m,1H), 2.21-2.12 (m, 4H), 1.76-1.61 (m, 6H), 1.45-1.42 (m, 1H), 1.32-1.14(m, 4H), 1.05-0.95 (m, 9H), 0.91 (m, 3).

The carboxylic acid 22D (18.5 mg, 0.029 mmol) stirred with CDI (6.0 mg)in DMF (1.5 mL) at 80° C. for 10 minutes. The reaction was cooled toroom temperature and compound 24E in DMF (0.15 mL) with DBU (4 eq.) wasadded and the reaction was heated in an 80° C. bath for 20 minutes. Thereaction was purified directly by reverse phase chromatography to yield7.6 mg of Compound No. 115. LCMS (M+1=745.2), ¹H-NMR (500 MHz,methanol-d₄): 9.30 (s, 0.5H), 8.02 (m, 0.5H), 7.71 (m, 1H), 7.60 (dt,J=7.2, 1.3 Hz, 1H), 7.46-7.41 (m, 2H), 5.86-5.79 (m, 1H), 5.35-5.28 (m,1H), 5.12-5.10 (m, 1H), 4.65 (m, 1H), 4.42 (dd, J=6.9, 10.6 Hz, 1H),4.28 (d, J=11.3 Hz, 1H), 3.95 (d, J=11.4 Hz, 1H), 3.62-3.47 (m, 2H),2.51-2.47 (m, 1H), 2.35-2.31 (m, 1H), 2.25-2.12 (m, 4H), 1.89 (dd,J=5.4, 8.1 Hz, 1H), 1.80-1.64 (m, 6H), 1.45-1.39 (m, 1H), 1.33-1.15 (m,3H), 1.04-0.97 (m, 11H), 0.73-0.57 (m, 4H).

Listed below in Table 9 are some physical data of exemplary compounds ofFormula I.

LC/MS data were acquired using the following:

-   Mass spectrometers: PESciex API-150-EX or Waters/Micromass ZQ or    Waters/Micromass Quattro II, or Waters/Micromass ZMD; Pumps:    Shimadzu LC-8A or Agilent 1100;-   Autosamplers: Gilson 215 or Gilson 819.

The following methods were used 3.0 mL/min flow rate, 10-99% CH₃CN(0.035% TFA)/H2O (0.05% TFA) gradient, Phenomenex Luna 5 m C18 column(50×4.60 mm); 1.5 mL/min flow rate, 10-90% CH₃CN (0.2% Formic acid)/H2O(0.2% Formic Acid) in 3 minutes, YMC-Pack Pro-C18 column (50×4.6 mm);1.0 mL/min flow rate, 10-90% CH3CN (0.2% Formic acid)/H2O (0.2% FormicAcid) in 5 minutes, YMC-Pro-C18 column (50×2.0 mm); 1.5 mL/min flowrate, 10-90% CH₃CN (0.1% TFA))/H2O (0.1TFA) in 3 minutes, YMC-PackPro-C18 column (50×4.60 mm)

TABLE 9 Physical data for Exemplary Compounds of Formula I. Com- FIA-FIA- pound LCMS LCMS MS MS No. (M + 1) RT (M − 1) (M + 1) NMR 1 754.4756.1 2 700.2 3.99 3 698.8 3.84 4 763 765 5 686.3 2.8 6 760.9 2.4 7710.2 3.35 8 707.9 9 638 640 10 724 726.1 11 662.4 664.3 12 602 2.94 13668.2 3.6 14 580.9 15 725.1 726.1 16 691.8 17 791.8 2.29 18 752.4 3.7419 696.3 3.7 H-NMR (500 MHz, CDCl₃)7.61 (d, J = 1.7 Hz, H), 7.51 (d, J =7.7 Hz, H), 7.38 (d, J = 8.2 Hz, H), 7.33 (t, J = 7.8 Hz, H), 7.26 (s,CDCl3), 7.16 (t, J = 6.5 Hz, H), 6.91 (d, J = 3.3 Hz, H), 6.55-6.50 (m,H), 5.35 (dd, J = 4.1, 8.5 Hz, H), 4.77-4.74 (m, H), 4.66 (d, J = 9.4Hz, H), 4.29 (d, J = 11.1 Hz, H), 3.71 (d, J = 11.2 Hz, H), 3.47 (d,1H), 3.29 (d, 1H), 2.78 (td, J = 7.3, 3.7 Hz, H), 2.63-2.59 (m, H),2.52-2.49 (m, H), 2.07- 2.30 (m, 2H), 1.98-1.92 (m, H), 1.77-1.61 (m,H), 1.47-1.40 (m, H), 1.35-1.25 (m, H), 1.21 (dd, J = 6.6, 12.5 Hz, H),1.01 (s, 9H), 0.98- 0.83 (m, H), 0.61-0.51 (m, H), 0.39 (t, J = 4.8 Hz,H) ppm 20 672 3.24 21 697.9 699.8 22 713 3.9 CDCl₃; 7.77 (d, 2H), 7.58(m, 4H), 7.41 (t, 2H), 7.32 (3H) 23 710 4.5 24 630.3 632.2 25 684.6686.5 26 761.4 3.23 27 672.4 3.7 28 675.3 3.44 29 698.361 3.8 30 767.63.15 31 701.3 2.74 32 730.3 732.1 33 685 3.5 34 559 35 706.1 3.66 36675.5 677.3 37 712 3.19 38 748.1 3.86 39 728 3.82 40 655.3 657.2 41 7293.4 42 701.3 2.75 43 654.1 656.1 44 658 3 45 720.2 3.29 46 670 3.78 47734.2 736.1 48 728.2 3.47 49 784.2 3.71 50 696.6 3.76 51 548.1 550 52704 53 696 2.04 54 634 3.24 55 642.3 644.2 56 600.6 602.4 57 755 58778.9 2.38 59 730.2 3.58 60 734.4 3.67 61 591.8 2.6 62 690.2 3.64 63 6983.83 64 595 65 634.4 1.8 66 734.4 736.2 67 720.4 722.2 68 675.2 2.6 69817.7 3.97 70 712.4 714.3 71 722.1 3.59 72 670 3.53 73 800.7 3.97 74 682684 75 713.1 2.8 76 643 3.09 77 734.2 3.92 78 79 684.2 3.7 80 553.1 55581 687 2.1 82 611 612.6 83 772.1 774 84 780.9 4.39 85 650 3.26 86 676.5678.3 87 712 3.2 88 666 3.31 89 695.2 3.53 90 758 759.9 91 848.7 2.3 92686 3.05 93 670.4 3.56 94 660 2.69 95 583.9 585.7 96 759.3 4.02 97 670.1672.1 98 602.6 1.63 99 726.7 2.4 100 704 3.64 101 666.1 668.3 102 710.23.6 103 657 3.2 104 690 3.24 105 682.1 684.2 106 690.1 691.9 107 6593.22 108 700 3.32 109 706.4 708.4 110 754 3.6 111 652.1 654 112 684 113700.7 2.3 114 712.4 714.3 115 745.2 3.73 116 716.3 718 117 653 118 726.43.6 119 691.8 120 811.5 3.95 121 714 3.27 122 711 3.42 123 631 3.09 124686.2 3.4 125 732.2 3.7 126 680.55 682.4 127 794.7 4.11 128 774 3.28 129758.2 3.78 130 690.2 3.48 131 720 3.53 132 711.4 3.5 133 728.6 3.8 134726.2 3.7 135 773.1 3.5 136 666 668.1 137 708.2 710.1 138 843.9 2.2 139708 3.35 140 672.7 2.13 141 792.7 4.13 142 676.2 677.9 143 700.7 2.32144 627.4 3.43 145 707.8 146 635 2.47 147 690 691.9 148 813.9 2.15 149606.8 150 713.5 715.2 151 706 707.8 152 688.4 3.2 153 714.3 3.2 154700.1 701.9 155 709.6 3.59 156 658 3.35 157 650 3.32 158 714.3 3.72 159688.3 690.3 160 613.5 615.4 161 769.1 3.5 162 730.2 3.7 163 708 164630.1 632.1 165 715.2 3.73 166 710 3.46 167 709.4 2.2 168 656.3 3.46 169702.2 3.71 170 754.2 3.96 171 613.1 172 717.1 173 667 3.17 174 642.3644.3 175 701.2 2.76 176 817.7 4.1 177 787 789 178 576.1 578.1 179 718.23.55 180 770.1 772.1 181 801.7 2.06 182 686.7 2.19 183 714 715.9 184672.2 2.96 185 724.2 3.6 186 755.3 3.41 187 708 3.53 188 698 3.76 189722 3.5 190 718.1 3.68 191 710.4 712.4 192 683.3 685.3 193 646 3.44 702703.7 194 672.1 674 195 700.2 3.59 196 672.1 674 197 722 3.39 198 723.8725.7 199 766.2 768.2 200 746.2 748.1 201 567.1 568.8 202 638 640 203730.3 3.7 204 734.2 3.85 205 637 3.35 206 871.9 3.96 207 576 2.87 208718.2 719.95 209 716.3 718.1 210 675 3.5 211 698.2 3.47 212 692.6 694.5213 748.2 3.6 214 769.3 770.9 215 733.3 735.3 216 767.1 3.1 217 670 3.6218 657 3 219 670.2 3.65 220 504.1 506 221 792.9 4.1 222 593 594.9 223678 3.42 224 707.9 710.1 225 795 226 698 3.89 227 558.5 2.91 228 670 229649.7 2.55 230 704.3 3.7 231 684 3.73 232 718.1 3.82 233 700.34 3.31H-NMR (500 MHz, CDCl₃): 7.61 (t, J = 1.6 Hz, 1H), 7.53 (dd, J = 1.2,7.6 Hz, 1H), 7.39 (dd, J = 1.7, 6.9 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H),7.26 (s, CDCl3), 7.16 (d, J = 7.3 Hz, 1H), 6.91 (d, J = 3.4 Hz, 1H),6.62 (d, J = 9.2 Hz, 1H), 5.35 (d, J = 4.1 Hz, 1H), 4.76 (t, J = 7.8 Hz,1H), 4.64 (d, J = 9.3 Hz, 1H), 4.29 (d, J = 10.8 Hz, 1H), 3.71 (d, J =11.2 Hz, 1H), 3.29-3.49 (dd, J = 2H), 2.78 (m, 1H), 2.63-2.59 (m, 1H),2.53- 2.51 (m, 1H), 2.37 (d, J = 2.2 Hz, H), 1.90- 1.96 (m, 1H),1.68-1.60 (m, H), 1.47-1.40 (m, H), 1.21-1.32 (m), 0.99 (s, 9H),0.95-0.83 (m, H), 0.60 (dd, J = 3.4, 9.5 Hz, H) ppm 234 722 724 235626.1 3.34 236 716.1 718.2 237 696.2 2.04 238 784.2 3.77 239 738 3.8 240668.2 670.2 241 713.4 2.94 242 667.4 669.4 243 605.9 2.77 244 653.2 245682 3.58 246 664.7 3.39 247 645.3 3.3 248 707.4 3.7 249 665 3.49 250714.2 3.8 251 613.4 615.2 252 682 3.7 253 672.3 3.72 254 728.4 3.6 255778.9 2.37 256 668.5 3.6 257 626 628 258 566.3 568.1 259 783 3.24 260662 3.16 261 821 3.21 262 612 3.4 263 608.9 611 264 692 3.31 265 698.33.87 266 704.3 706.2 267 575.6 577.4 268 752 3.87 269 617.6 2.71 270 720271 704 3.61 272 696 698.1 273 644 3.46 274 714.1 716.2 275 686.7 2.21276 716.1 717.8 277 728.3 3.75 278 782 3.9 279 670.3 672.2 280 638.2 640281 684.5 686.4 282 772.2 3.19 283 645 2.02 284 698 3.8 285 695.7 697.6286 760 3.13 287 708 3.4 288 695 3.46 289 786.2 3.7 290 695.7 697.6 291787 3.02 292 685.4 3.36 293 711.1 3.7 294 684 3.76 295 654.5 656.5 296748.7 2.37 297 718 3.48 298 800.6 2.3 299 706.6 708.3 300 714.7 2.36 301692.1 694.1 302 817.9 3.96 303 651.5 2.9 304 727 2.87 305 726.2 3.52 306757.5 759.5 307 744 3.57 308 702.5 3.4 309 715.3 3.59 310 683 685 311767.2 3.4 312 789.1 791 313 640 3.1 314 718 720 315 755.3 3.67 316 813815 317 747.2 3.35 318 644.1 645.9 319 658 3.57 320 775.3 777.1 321744.3 3.75 322 666 323 746.3 324 716.9 3.5 325 676.5 326 654.1 655.9 327686.5 688.4 328 746.7 3.65 329 613.4 615.2 330 678 331 726.5 728.3 332694.5 1.82 333 707 3.43 334 705.8 3.66 335 720 3.5 336 774.2 3.8 337680.6 682.6 338 736.2 3.6 339 757 3.24 340 682 684 341 697.1 2.86 342626 2.3 343 559 344 684.2 345 696 3.98 346 710.2 3.52 347 742.7 2.28 348531.6 533.3 349 730.4 3.5 350 700.3 3.94 351 634.5 636.3 352 675.6 677.3353 691.9 354 700.6 702.5 355 762.2 3.91 356 760 3.11 357 695 2.24 358686.1 687.9 359 732.4 3.12 360 698 3.87 361 698 3.83 362 652 654 363 722724.1 364 701.9 3.21 365 676.1 366 728.1 3.6 367 636 2.8 368 770.2 3.64369 721.1 723.2 370 728.3 3.62 371 695 3.7 372 712.3 3.98 373 723.4 2.3374 688.4 3.2 375 656 658 376 636.3 3.36 377 795.4 3.29 378 761.1 763379 645 3.16 380 649.5 1.97 381 731.4 3.3 382 758.3 760.1 383 612.1 3.2384 629.4 2.78 385 504.1 506 386 504.1 506 387 716 3.13 388 656 3.42 389723.4 2.3 390 696.1 698 391 583 584.8 392 660 3.05 393 696 4.05 394703.351 3.3 395 730 3.55 396 698.25 3.89 397 650.5 1.73 398 696.1 698399 742.7 2.16 400 583 584.8 401 698.3 3.9 402 764.1 3.4 403 716.9 719404 702.5 2.07 405 406 670.1 672.2 407 632 3.35 408 735.7 3.14 409 410714.5 2.12 411 566.10 568 412 652 3 413 717.9 719.8 414 769.1 771 415639.5 641.5 416 734.5 3.83 417 653.3 418 686.3 3.1 419 714 3.26 420 7043.56 421 591.6 1.87 422 693.4 695.4 423 714.2 716.1 424 706 3.31 425691.8 693.8 426 714.2 3.48 427 666 3.38 428 702.1 704 429 760.9 2.38 430753.2 3.86 431 691.9 3.3 432 645 3.05 433 835.7 4.16 434 720 3.5 435 7443.56 436 774 776.2 437 730.2 3.7 438 548.2 549.9 439 603.4 3.34 440602.6 604.4 441 724.9 2.22 442 712.6 714.5 443 615.6 3.25 444 676.2678.2 445 742.35 3.2 446 756.2 3.68 447 749.7 1.78 448 608.1 610 449 756351 450 698.3 700.2 451 630 452 700.3 3.94 453 694.3 3.64 454 761.1 3.3455 724.4 456 710.5 712.2 457 602.4 3.12 458 803.7 3.97 459 684.2 3.6460 587.5 3.01 461 735.7 1.8 462 610.1 611.9 463 708.4 3.7 464 706.1708.2 465 740.4 742.2 466 738.6 3.63 467 696.345 3.7 468 686.2 2.98 469681.3 3.39 470 610.1 612.05 471 708.2 3.5 472 837 839.1 473 706 708.1474 710.2 3.2 475 714 3.3 476 734.4 3.67 477 717.37 3.3 478 690.2 3.73479 690.2 3.66 480 672 673.9 481 718 720 482 698.2 700.1 483 734.6 1.87484 660 1.44 485 676 3.38 486 803.6 805.4 487 762 3.26 488 794.7 4.07489 716.5 3.59 490 709.4 711.4 491 754 492 659 3.39 734.4 736.2 493658.3 3.61 494 744.2 3.71 495 688.2 3.3 496 698.3 3.83 497 694 2.16 498670.3 672.2 499 726.2 3.65 500 694.3 3.64 501 720.5 3.62 502 724.1 725.9503 700 3.36 504 692.8 2.13 505 713.8 2.73 506 718 1.87 507 854.7 4.15508 686.7 2.21 509 724.3 510 756.2 2.95 511 680.5 682.54 512 746.4 748.3513 726.4 728.2 514 635 3.68 515 688.4 3.2 516 700 2.98 517 744.1 746.1518 775.2 3.3 519 636 520 660 3.5 521 720.1 3.84 522 670 3.59 523 6723.1 524 525 735.2 737 526 694 3.64 527 746.1 748.1 528 731.9 3.38 529732 2.89 530 722 531 650 3.46 532 644 3.39 533 694 696 534 730.5 3.67535 668.2 3 536 705.8 707.9 537 742.7 2.25 538 731.2 3.7 539 743.2 744.2540 778.9 4.15 541 700.34 3.2 542 685.34 3.5 543 695.7 697.7 544 746.22.3 545 696.1 697.9 546 748.7 2.38 547 726.4 728.25 548 682 684 549 696697.9 550 653.3 654 551 609.3 552 692.3 3.51 553 712.2 2.6 554 670.5 2.9555 556 557 725.8 3.4 558 679 3.46 559 702 704 560 696.2 698 561 730.43.7 562 716 3.41 563 695 2.46 564 707.9 565 762.2 3.55 566 628 630 567774.7 3.19 568 712.7 2.3 569 671.9 570 656.1 658.2 571 730.2 3.4 572639.1 641.2 573 694.1 574 634.5 1.7 575 714.4 3.1 576 680 2.2 577 7183.51 578 680.5 682.4 579 698 3.72 580 597 2.87 581 720 3.51 582 714.43.6 583 693 3.35 584 744 585 762 3.68 586 707 3.2 587 730.5 3.68 588745.7 4.09 589 735.20 3.70 590 694.30 3.65 591 651.30 3.26 592 651.303.24 593 685.20 3.53 594 700.20 3.72VI. Assays for Detecting and Measuring Inhibition Properties ofCompounds

A. HCV Enzyme Assays

1. Construction and Expression of the HCV NS3 Serine Protease Domain

A DNA fragment encoding residues Ala¹-Ser¹⁸¹ of the HCV NS3 protease(GenBank CAB46913) was obtained by PCR from the HCV Con1 repliconplasmid, I₃₇₇meo/NS3-3′/wt (re-named as pBR322-HCV-Neo in this study)[V. Lohmann et al., Science, 285, pp. 110-113 (1999)] and inserted intopBEV11 (S. Chamber, et al., personal communication) for expression ofthe HCV proteins with a C-terminal hexa-histidine tag in E. coli. Allconstructs were confirmed by sequencing.

The expression constructs for the HCV NS3 serine protease domain wastransformed into BL21/DE3 pLysS E. coli cells (Stratagene). Freshlytransformed cells were grown at 37° C. in a BHI medium (DifcoLaboratories) supplemented with 100 μg/ml carbenicillin and 35 μg/mlchloramphenicol to an optical density of 0.75 at 600 nm Induction with 1mM IPTG was performed for four hours at 24° C. The cell paste washarvested by centrifugation and flash frozen at −80° C. prior to proteinpurification. All purification steps were performed at 4° C. Next, 100 gof cell paste was lysed in 1.5 L of buffer A (50 mM HEPES (pH 8.0), 300mM NaCl, 0.1% n-octyl-β-D-glucopyranoside, 5 mM β-mercaptoethanol, 10%(v/v) glycerol) and stirred for 30 minutes. The lysate was homogenizedusing a Microfluidizer (Microfluidics, Newton, Mass.), followed byultra-centrifugation at 54,000×g for 45 minutes Imidazole was added tothe supernatant to a final concentration of 5 mM along with 2 mL ofNi-NTA resin pre-equilibrated with buffer A containing 5 mM imidazole.The mixture was rocked for three hours and washed with 20 column volumesof buffer A plus 5 mM imidazole. The HCV NS3 protein was eluted inbuffer A containing 300 mM imidazole. The eluate was concentrated andloaded onto a Hi-Load 16/60 Superdex 200 column, pre-equilibrated withbuffer A. The appropriate fractions of the purified HCV protein werepooled and stored at −80° C.

2. HCV NS3 Protease Domain Peptide Cleavage Assay

This assay is a modification of that described by Landro, et al. (LandroJ A, Raybuck S A, Luong Y C, O'Malley E T, Harbeson S L, Morgenstern KA, Rao G and Livingston D L. Biochemistry 1997, 36, 9340-9348), and usesa peptide substrate (NS5AB), based on the NS5A/NS5B cleavage site forgenotype 1a HCV. The substrate stock solution (25 mM) was prepared inDMSO containing 0.2 M DTT and stored at −20° C. A synthetic peptidecofactor (KK4A) was used as a substitute for the central core region ofNS4A. Peptide sequences are shown in the table below. The reaction wasperformed in a 96-well microtiter plate format using 25 ηM to 50 ηM HCVNS3 protease domain in buffer containing 50 mM HEPES pH 7.8, 100 mMNaCl, 20% glycerol, 5 mM DTT and 25 μM KK4A. The final DMSOconcentration was no greater than 2% v/v. Reactions were quenched byaddition of trifluoroacetic acid (TFA) to yield a final concentration of2.5%.

Peptide Sequences Used with HCV NS3 Protease Domain Peptide SequenceNS5AB NH₂-EDVV-(alpha)Abu-CSMSY-COOH [SEQ ID NO: 2] KK4ANH₂-KKGSVVIVGRIVLSGK-COOH [SEQ ID NO: 3]

The SMSY product was separated from substrate and KK4A using a microboreseparation method. The instrument used was a Agilent 1100 with a G1322Adegasser, either a G1312A binary pump or a G1311A quaternary pump, aG1313A autosampler, a G1316A column thermostated chamber and a G1315Adiode array detector. The column was a Phenomenex Jupiter, 5 μm C18, 300{acute over (Å)}, 150×2 mm, P/O 00E-4053-B0, with a flow-rate of 0.2mL/min The column thermostat was at 40° C. Mobile phases were HPLC gradeH₂O/0.1% TFA (solvent A) and HPLC grade CH₃CN/0.1% TFA (solvent B). TheSMSY product peak was quantitated using the data collected at 210 ηM.

3. Construction and Expression of NS3•4A Protease

Using standard recombinant DNA techniques, a cDNA fragment encoding thesequence for NS3 and NS4A, residues Ala₁₀₂₇ to Cys₁₇₁₁ from the HCVsub-type strain la, containing an N-terminal hexa-histidine sequence,was cloned into the baculoviral transfer vector pVL1392 (Webb N R andSummers M D (1990) Expression of proteins using recombinantbaculoviruses, Techniques 2:173-188). Recombinant baculovirus containingNS3•4A was produced by co-transfection of pVL1392-His-NS3•4A withlinearized Autographa californica nuclear polyhedrosis virus (AcMNPV)DNA into Spodoptera frugoperda (Sf9) insect cells. The transfectedinsect cells containing recombinant baculovirus clones were subsequentlyisolated by plaque purification. High-titer clonal baculovirus wasroutinely used to infect Sf9 insect cells for protein production. Inproduction, Sf9 cells were grown at 27° C. until they reached a densityof 2.0-×10⁶ cells/ml. At this point, the insect cells were infected withvirus. After 72 hours or when the cell viability was between 70-80% theculture was harvested and the cells were ready for purification.

4. Purification of NS3•4A Protein

The NS3•4A protein (SEQ ID NO:1) was purified as follows. Cell paste wasthawed in at least five volumes of Lysis Buffer (50 mM Na₂HPO₄ pH 8.0,10% Glycerol, 300 mM NaCl, 5 mM β-mercaptoethanol, 0.2 mM PMSF, 2.5μg/ml Leupeptin, 1.0 μg/ml E64, 2.0 μg/ml Pepstatin) per gram of cellpaste. The cell paste was then homogenized on ice using a Douncehomogenizer. The cells were next mechanically disrupted by passing oncethrough a microfluidizer (Microfluidics Corporation, Newton, Mass.), andthe cell lysate was collected on ice. The cell lysates was centrifugedat 100,000×g for 30 minutes at 4° C. and the supernatants were decanted.Optionally, the pellets were resuspended in wash buffer (LysisBuffer+0.1% β-octyl glucopyranoside), homogenized using a Douncehomogenizer and centrifuged at 100,000×g for 30 minutes at 4° C.Insoluble NS3•4A was extracted from the pellets by resuspending inExtraction Buffer (Lysis Buffer+0.5% lauryl maltoside) using 2.5 ml/gcell paste. The mixture was homogenized using a Dounce homogenizer andmixed at 4° C. for three hours or more. The mixture was centrifuged at100,000×g for 30 minutes at 4° C. The supernatants were decanted andpooled.

The NS3•4A protein was further purified using Nickel-NTA metal affinitychromatography. Imidazole from a 2 M stock, pH 8.0, solution was addedto the pooled supernatants so that the final concentration of imidazolewas 10 mM. The supernatants were incubated batchwise overnight at 4° C.with Nickel-NTA affinity resin that had been pre-equilibrated with LysisBuffer+10 mM imidazole. 1 ml of resin per 5 μg of expected NS3-4A wasused. The resin was next settled by gravity or by centrifugation at500×g for five minutes. The resin was next poured into a gravity flowcolumn and washed with 10 or more column volumes of Nickel Wash Buffer(Lysis Buffer+0.1% lauryl maltoside+10 mM imidazole). The column wasnext eluted with three to four column volumes of Nickel Elution Buffer(Nickel Wash Buffer+300 mM imidazole). The elution fractions werecollected on ice and evaluated using SDS-PAGE. To prevent NS3-4Aproteolysis, 100 μM DFP protease inhibitor was added to gel samplesbefore adding SDS sample buffer and boiling. The peak fractions werepooled and protein concentration was determined by measuring absorbanceat 280 ηm and by dividing by the extinction coefficient (e), which forNS3•4A is 1.01.

The NS3•4A was purified further using gel filtration chromatography. ASuperdex 200 26/60 column was equilibrated with Superdex Buffer (20 mMHEPES pH 8.0, 10% glycerol, 300 mM NaCl, 10 mM β-mercaptoethanol, 0.05%lauryl maltoside) at a rate of 3 ml/min The nickel purified NS3•4A wasconcentrated in a Centriprep 30 to greater than 2 mg/ml, if necessary,and was filtered through a 0.2 μm syringe filter and up to 10 ml wasloaded onto the Superdex 200 column After 0.3 column volumes passedthrough, 4-5 ml fractions were collected. Fractions were evaluated bySDS-PAGE. NS3•4A protein elutes in two peaks. Peak 1 contains aggregatedNS3•4A and peak 2 contains active protein. The fractions of peak 2 werepooled, aliquoted and frozen at −70° C.

Analysis of NS3•4A Protein

ANALYSIS ENTIRE PROTEIN Length 695 amino acids Molecular Weight74,347.78 1 microgram 13.450 picot moles Molar Extinction Coefficient73430 1 A₂₈₀ corresponds to 1.01 mg/ml Isoelectric Point 6.50 Charge atpH 7 −3.58

5. HCV NS3 Peptide Cleavage Assay

This assay follows the cleavage of a peptide substrate by full-lengthhepatitis C viral protein NS3•4A. One of three peptide substrates basedon the NS5A/NS5B cleavage site for genotype 1a HCV is used to measureenzyme activity. All substrate stock solutions (25 mM) were prepared inDMSO containing 0.2M DTT and stored at −20° C. A synthetic peptidecofactor (NS4A Peptide) was used to supplement NS4A. Peptide sequencesare shown below. The hydrolysis reaction was performed in a 96-wellmicrotiter plate format using 100 M to 125 ηM HCV NS3•4A in buffercontaining 50 mM HEPES pH 7.8, 100 mM NaCl, 20% glycerol, 5 mM DTT and25 μM NS4A Peptide. The final DMSO concentration was no greater than 2%v/v. Reactions using NS5AB or NS5AB-EDANS as substrate were quenched bythe addition of 10% trifluoroacetic acid (TFA) to yield a final TFAconcentration of 2.5%. Reactions using FITC-NS5AB-1 as substrate werequenched by the addition of 0.4M formic acid to yield a finalconcentration of 0.08M acid.

Enzymatic activity was assessed by separation of substrate and productsby reverse phase HPLC. The instrument used was a Agilent 1100 with aG1322A degasser, either a G1312A binary pump or a G1311A quaternarypump, a G1313A autosampler, a G1316A column thermostated chamber, aG1321A fluorescence detector and a G1315A diode array detector. Thecolumn thermostat was at 40° C. For substrate NS5AB the column was aPhenomenex Jupiter, 5 μm C18, 300 {acute over (Å)}, 150×2 mm, P/000E-4053-B0, with a flow-rate of 0.2 mL/min using HPLC grade H₂O/0.1%TFA (solvent A) and HPLC grade CH₃CN/0.1% TFA (solvent B) as mobilephases. The C-terminal product peak (NH2-SMSY —COOH) was quantitatedusing the absorbance data collected at 210 ηm. For substrate NS5AB-EDANSthe column was a Phenomenex Aqua, 5 μm C18, 125 {acute over (Å)}, 50×4.6mm, P/O 00B-4299-E0, with a flow-rate of 1.0 mL/min using HPLC gradeH₂O/0.1% TFA (solvent A) and HPLC grade CH₃CN/0.1% TFA (solvent B) asmobile phases. The C-terminal product peak(NH2-SMSYT-Asp(EDANS)-KKK-COOH) was quantitated using the fluorescencedata collected at 350 ηm excitation/490 ηm emission. For substrateFITC-NS5AB-1 the column was a Phenomenex Prodigy, 5 μm ODS(2), 125{acute over (Å)}, 50×4.6 mm, P/O 00B-3300-E0, with a flow-rate of 1.0mL/min using 10 mM sodium phosphate pH 7.0 in HPLC grade H2O (solvent A)and 65% HPLC Grade CH₃CN/35% 10 mM sodium phosphate pH 7.0 in HPLC gradeH2O (solvent B) as mobile phases. The N-terminal product peak(FITC-Ahx-EDVV-(alpha)Abu-C—COOH) was quantitated using the fluorescencedata collected at 440 nm excitation/520 nm emission. Alternatively, theratio of N-terminal product to unreacted FITC-NS5AB-1 substrate wasdetermined using a Caliper LabChip 3000 with detection at 488 nmexcitation/530 nm emission, using a chip buffer of 100 mM Tris pH 7.0,10 mM EDTA, 0.01% (v/v) Brij-35, and 0.1% (v/v) CR-3.

Peptide Sequences used with HCV NS3.

Peptide Sequence NS4A Peptide NH₂-KKGSVVIVGRIVLSGKPAIIPKK-COOH[SEQ ID NO: 4] NS5AB NH₂-EDVV-(alpha)Abu-CSMSY-COOH [SEQ ID NO: 2]NS5AB-EDANS NH₂-EDVV-(alpha)Abu-CSMSYT-Asp(EDANS)-KKK-COOH [SEQ ID NO: 5] FITC-NS5AB-1FITC-Ahx-EDVV-(alpha)Abu-CSMSYTKK-NH₂ [SEQ ID NO: 6]

6. Determination of Km and Vmax

To determine the kinetic parameters Km and Vmax, the HCV NS3 proteasedomain or HCV NS3•4A was reacted with peptide substrate under the assayconditions described above. Peptide substrate concentration was variedbetween 3 μM and 200 μM, with less than 20 percent conversion at allsubstrate concentrations. The ratio of the product peak area (asdetermined by reverse phase HPLC) to the reaction time yielded a rate ofenzyme catalyzed hydrolysis. These rate vs. substrate concentration datapoints were fit to the Michaelis-Menten equation using non-linearregression. The value of k_(cat) was determined from Vmax using thenominal protease concentration and a fully cleaved substrate peptide asan instrument calibration standard.

Kinetic Parameters for Peptide Substrates with HCV NS3 or NS3 ProteaseDomain

k_(cat)/Km Enzyme Substrate Km (μM) (M⁻¹sec⁻¹) NS3 Protease NS5AB 25 3.0× 10⁴ Domain NS3.4A NS5AB 30 7.9 × 10³ NS3.4A NS5AB-EDANS 56 1.4 × 10³NS3.4A FITC-NS5AB-1 15 1.2 × 10³

7. Determination of Compound Potency

To evaluate apparent Ki values, all components except the test compoundand substrate were pre-incubated for 5-10 minutes at room temperature.Then, test compound, dissolved in DMSO, was added to the mixture andincubated for either 15 minutes or 60 minutes at 30° C. Neat DMSO wasincluded as a no inhibitor control. The cleavage reaction was initiatedby the addition of peptide substrate at a concentration either equal toKm or equal to one-half times Km, and allowed to proceed at 30° C. fortwenty minutes. At the end of the reaction the mixture was quenched, andthe extent of reaction was determined as described above. Elevenconcentrations of compound were used to titrate enzyme activity forinhibition. Activity vs. inhibitor concentration data points were fit tothe Morrison equation describing competitive tight-binding enzymeinhibition using non-linear regression (Sculley M J and Morrison J F.Biochim Biophys. Acta. 1986, 874, 44-53).

The tested compounds of formula I generally exhibited Ki values fromabout 0.008 to about 20 μM. In some embodiments, the compounds offormula I exhibited Ki values from about 0.008 to about 0.100 μM. Insome other embodiments, the compounds of formula I exhibited Ki valuesfrom about 0.100 to about 0.500 μM. In still some other embodiments, thecompounds of formula I exhibited Ki values from 0.500 to about 5.000 μM.

Examples of activities of the compounds of formulae (I, Ia, and Ib) oninhibiting serine protease receptors are shown below in Table 10. Forcompound activities for serine protease measured using the HCV EnzymeAssays, serine protease activity is illustrated with “+++” if activitywas measured to be less than 0.1 μM, “++” if activity was measured to befrom 0.1 μM to 0.5 μM, “+” if activity was measured to be greater than0.5 μM, and “−” if no data was available. It should be noted that 0%efficacy is the minimum response obtained with the DMSO only control.The Enzyme Assay 1 refers to the HCV NS3 Protease Domain PeptideCleavage Assay and Enzyme Assay 2 refers to the HCV NS3 Peptide CleavageAssay.

TABLE 10 HCV Enzymatic Assay Activities and efficacies of exemplarycompounds in accordance to Formulae I. Compound Enzyme Enzyme No. Assay1 Assay 2 1 − − 2 − − 3 ++ +++ 4 ++ 5 +++ +++ 6 + + 7 + − 8 + − 9 + −10 + + 11 + − 12 ++ +++ 13 +++ +++ 14 + − 15 ++ − 16 + − 17 + ++ 18 ++++++ 19 ++ +++ 20 + − 21 + + 22 − − 23 +++ ++ 24 ++ − 25 + − 26 ++ +++ 27++ − 28 + ++ 29 ++ ++ 30 ++ +++ 31 − ++ 32 ++ − 33 +++ − 34 + − 35 − −36 ++ − 37 + − 38 − ++ 39 ++ − 40 + − 41 +++ − 42 ++ +++ 43 ++ +++ 44 ++− 45 + ++ 46 + + 47 + − 48 − − 49 − +++ 50 − +++ 51 + − 52 ++ − 53 + −54 + ++ 55 ++ − 56 ++ − 57 − − 58 + + 59 − +++ 60 ++ ++ 61 + − 62 ++ ++63 + + 64 + − 65 + + 66 ++ − 67 +++ +++ 68 − +++ 69 + ++ 70 + − 71 + −72 ++ − 73 + ++ 74 ++ +++ 75 ++ − 76 ++ − 77 ++ +++ 78 ++ − 79 ++ +++80 + − 81 + − 82 ++ − 83 ++ − 84 + + 85 ++ − 86 ++ − 87 ++ ++ 88 + ++ 89− ++ 90 − +++ 91 + + 92 ++ − 93 ++ +++ 94 +++ − 95 ++ − 96 + ++ 97 − +++98 + ++ 99 + + 100 + + 101 + − 102 ++ − 103 ++ − 104 − +++ 105 − +++106 + − 107 ++ − 108 ++ − 109 +++ − 110 ++ − 111 ++ ++ 112 ++ +++113 + + 114 +++ +++ 115 + ++ 116 + − 117 − ++ 118 + − 119 ++ ++ 120 + +121 ++ +++ 122 ++ − 123 +++ − 124 + ++ 125 ++ ++ 126 ++ ++ 127 ++ ++ 128++ − 129 − +++ 130 ++ +++ 131 − +++ 132 ++ ++ 133 ++ ++ 134 ++ +++ 135 +− 136 ++ ++ 137 − − 138 + + 139 + − 140 ++ +++ 141 + ++ 142 + − 143 ++++ 144 + + 145 ++ − 146 + − 147 +++ − 148 + + 149 + − 150 ++ − 151 ++ −152 ++ − 153 ++ ++ 154 ++ − 155 + − 156 +++ − 157 + + 158 +++ +++ 159 ++− 160 + − 161 + − 162 ++ +++ 163 +++ +++ 164 + − 165 ++ +++ 166 ++ − 167++ − 168 ++ ++ 169 − ++ 170 − +++ 171 + + 172 + ++ 173 +++ − 174 ++ −175 − − 176 ++ ++ 177 + ++ 178 ++ ++ 179 ++ − 180 + − 181 + + 182 + +183 ++ − 184 − +++ 185 + ++ 186 + + 187 + − 188 + + 189 + − 190 ++ +++191 + − 192 − − 193 ++ − 194 + − 195 − +++ 196 ++ − 197 ++ − 198 + −199 + − 200 + − 201 ++ − 202 + − 203 ++ − 204 ++ ++ 205 + − 206 ++ ++207 + − 208 ++ − 209 ++ − 210 + − 211 − +++ 212 ++ +++ 213 + + 214 + −215 ++ − 216 + − 217 +++ − 218 + + 219 − +++ 220 + − 221 ++ ++ 222 + −223 ++ − 224 ++ − 225 − +++ 226 ++ − 227 + + 228 + − 229 + − 230 ++ −231 ++ − 232 − − 233 ++ +++ 234 ++ − 235 + − 236 + − 237 ++ − 238 − +239 ++ − 240 + − 241 + ++ 242 +++ − 243 + − 244 ++ − 245 ++ − 246 + +247 +++ − 248 + + 249 ++ − 250 − +++ 251 + − 252 ++ +++ 253 + + 254 ++ −255 ++ ++ 256 ++ − 257 ++ − 258 + − 259 ++ − 260 ++ − 261 ++ − 262 + −263 ++ +++ 264 − +++ 265 + ++ 266 ++ ++ 267 − − 268 ++ + 269 + + 270 ++− 271 ++ +++ 272 + + 273 ++ − 274 + − 275 ++ ++ 276 + − 277 + ++ 278 + −279 ++ − 280 + + 281 +++ − 282 + ++ 283 + − 284 ++ ++ 285 ++ − 286 − +++287 ++ − 288 ++ − 289 + ++ 290 ++ − 291 + − 292 ++ +++ 293 + + 294 ++ −295 ++ − 296 + + 297 − +++ 298 ++ ++ 299 + − 300 ++ ++ 301 ++ − 302 ++++ 303 + − 304 ++ +++ 305 − ++ 306 + − 307 ++ +++ 308 ++ − 309 + +310 + + 311 + − 312 +++ +++ 313 + − 314 ++ − 315 +++ +++ 316 ++ − 317 +− 318 ++ − 319 ++ − 320 + + 321 ++ +++ 322 + − 323 ++ − 324 + − 325 + −326 ++ ++ 327 + − 328 + + 329 + + 330 ++ − 331 + − 332 + ++ 333 ++ −334 + − 335 + − 336 + + 337 − +++ 338 + ++ 339 ++ +++ 340 ++ − 341 + −342 + − 343 + − 344 − − 345 + − 346 − +++ 347 + + 348 + − 349 ++ −350 + + 351 + + 352 + − 353 + − 354 + − 355 + + 356 − +++ 357 ++ − 358++ − 359 ++ +++ 360 + − 361 ++ +++ 362 ++ +++ 363 + − 364 + − 365 ++ −366 ++ +++ 367 + − 368 − ++ 369 − +++ 370 ++ +++ 371 ++ − 372 ++ ++ 373++ − 374 ++ − 375 + − 376 + + 377 ++ +++ 378 ++ − 379 +++ − 380 − + 381++ − 382 +++ − 383 + − 384 + + 385 + − 386 − − 387 ++ − 388 +++ +++389 + − 390 ++ +++ 391 ++ − 392 ++ − 393 ++ − 394 +++ − 395 + + 396 −+++ 397 + + 398 + − 399 + + 400 +++ − 401 ++ ++ 402 ++ − 403 + − 404 +++ 405 + − 406 − +++ 407 ++ − 408 + ++ 409 − − 410 + + 411 ++ +++ 412 ++++ 413 + − 414 + − 415 ++ − 416 + ++ 417 +++ − 418 ++ +++ 419 ++ ++420 + + 421 − + 422 ++ − 423 + − 424 ++ − 425 + − 426 + ++ 427 + − 428++ − 429 + + 430 ++ +++ 431 ++ − 432 + − 433 + + 434 ++ − 435 ++ − 436++ − 437 + + 438 + ++ 439 + + 440 − − 441 + ++ 442 ++ +++ 443 + + 444 ++− 445 ++ +++ 446 − +++ 447 + ++ 448 ++ − 449 ++ − 450 +++ − 451 + −452 + ++ 453 ++ − 454 ++ − 455 + − 456 ++ − 457 + + 458 + + 459 + ++460 + + 461 + ++ 462 ++ − 463 + − 464 + − 465 ++ − 466 + + 467 ++ +++468 − +++ 469 + ++ 470 − − 471 ++ − 472 + − 473 − − 474 + − 475 ++ − 476++ ++ 477 +++ − 478 ++ − 479 − − 480 + − 481 + − 482 + − 483 + + 484 ++− 485 − +++ 486 ++ − 487 ++ − 488 + ++ 489 + ++ 490 ++ − 491 + − 492 +++− 493 ++ +++ 494 +++ − 495 + − 496 + + 497 ++ − 498 ++ − 499 +++ +++ 500++ +++ 501 + + 502 − − 503 ++ − 504 ++ − 505 ++ − 506 + − 507 + + 508 ++++ 509 + − 510 ++ +++ 511 ++ − 512 ++ − 513 − +++ 514 + − 515 ++ − 516 +− 517 + − 518 +++ − 519 ++ − 520 + − 521 ++ +++ 522 − +++ 523 ++ − 524 +− 525 + − 526 ++ − 527 ++ − 528 ++ − 529 − +++ 530 + − 531 ++ ++ 532 + −533 + − 534 + + 535 ++ − 536 + − 537 + + 538 + − 539 ++ − 540 ++ ++ 541++ +++ 542 +++ − 543 + − 544 ++ − 545 ++ − 546 ++ ++ 547 +++ − 548 ++ −549 +++ − 550 ++ − 551 ++ − 552 ++ − 553 ++ − 554 + − 555 ++ − 556 − +++557 ++ − 558 ++ − 559 + + 560 ++ − 561 ++ − 562 − +++ 563 ++ − 564 + −565 + + 566 + + 567 + ++ 568 + + 569 +++ − 570 ++ − 571 ++ − 572 ++ −573 + + 574 + + 575 +++ +++ 576 ++ − 577 − +++ 578 + − 579 ++ − 580 ++ −581 − +++ 582 ++ − 583 ++ − 584 + − 585 ++ − 586 + + 587 + ++ 588 + +

B. HCV Cell Assays

Huh-7 cells were propagated in Dulbecco's modified Eagle's medium (DMEM,JRH Biosciences, Lenexa, Kansas) supplemented with 10% heat-inactivatedFBS (fetal bovine serum), 2 mM L-glutamine, and nonessential amino acids(JRH). The cells were transfected with an in vitro transcribed HCVreplicon RNA identical to replicon 1377neo/NS3-3′/wt as described byLohmann et al. (1999). Stable cell clones were selected and maintainedin the presence of 250 μg/mL G418 (Invitrogen, Carlsbad, Calif.). One ofthe clones, 24-2, was used in the subsequent HCV replicon assays. Thereplicon cells were propagated in DMEM supplemented with 10% FBS, 2 mML-glutamine, nonessential amino acids, and 250 μg/mL G418. The cellswere split twice per week in fresh media upon reaching confluence. Thereare approximately 200-300 copies of HCV RNA per replicon cell.

HCV replicon RNA from cells was measured using the Quantigene DiscoverXL kit (Panomics Inc., Fremont Calif.) as per the manufacturer'sinstructions. Briefly, compound-treated replicon cells were lysed andimmobilized on to capture plates using HCV specific oligonucleotidesover night and the relative amounts of captured RNA was measured usingoligonucleotide probe sets as per the manufacturer's instructions.

1. 2-Day HCV Replicon IC₅₀ Assay

On the day prior to the assay, 104 replicon cells were plated per wellof a 96-well plate and allowed to attach and grow overnight in DMEM(Invitrogen, Carlsbad, Calif.) supplemented with 10% heat-inactivatedFBS (JRH Biosciences, Lenexa, Kans.), 2 mM L-glutamine (Invitrogen),nonessential amino acids (Invitrogen) and 250 μg/ml G418 (Invitrogen).Compounds were serially diluted in DMEM plus 2% FBS and 0.5% DMSO (SigmaChemical Co., St. Louis, Mo.) without G418. HCV replicon RNA from cellswas measured using the Quantigene Discover XL kit (Panomics Inc.,Fremont Calif.) as per the manufacturer's instructions. Briefly,compound-treated replicon cells were lysed and immobilized on to captureplates using HCV specific oligonucleotides overnight and the relativeamounts of captured RNA was measured using oligonucleotide probe sets asper the manufacturer's instructions. Unless indicated otherwise, eachdata point represents the average of three replicates. The IC₅₀ is theconcentration of the compound at which the HCV replicon RNA level incells is reduced by 50% as compared to the untreated replicon cellcontrols. To monitor the effect of compounds on cell proliferation orcell viability, replicon cells were treated with serially dilutedcompounds for 48 h, after which cell viability was determined using aCellTiter Glo assay (Promega, Madison, Wis.). Each CC₅₀ is derived fromthree replicates and is the concentration of the compound at which thenumber of viable cells is reduced by 50% as compared to untreated cellcontrols. The IC₅₀ and CC₅₀ was determined using 4 parameter curvefitting in the SoftMax Pro program (Molecular Devices, Sunnyvale,Calif.).

2. 5-Day HCV Replicon IC₉₉ Assay

On the day prior to the assay, HCV replicon cells were plated at a lowdensity of 2500 cells per well in a 96-well plate so the cells would notreach confluence during 5 days in culture. Compounds were seriallydiluted in DMEM containing 10% FBS and 0.5% DMSO in the absence of G418.Fresh media and compounds were added to the cells on day 1 and day 3.After the cells were treated with antiviral compounds for 5 days, HCVreplicon RNA from cells was measured using the Quantigene Discover XLkit (Panomics Inc., Fremont Calif.) as per the manufacturer'sinstructions. Briefly, compound-treated replicon cells were lysed andimmobilized onto to capture plates using HCV specific oligonucleotidesovernight and the relative amounts of captured replicon RNA was measuredusing oligonucleotide probe sets (Panomics) as per manufacturer'sinstructions. Each data point represents the average of two replicates.The IC₉₉ is the concentration of the compound at which the HCV repliconRNA level in cells is reduced by 2 logs as compared to the untreatedcell controls. To monitor the effect of compounds on cell proliferationor cell viability, replicon cells were treated with serially dilutedcompounds for 5 days, after which cell viability was determined using aCellTiter Glo assay (Promega, Madison, Wis.). Each CC₅₀ is derived fromtwo replicates and is the concentration of the compound at which thenumber of viable cells is reduced by 50% as compared to untreated cellcontrols. The IC₉₉ and CC₅₀ were determined by 4 parameter curve fittingmethod using the Prism software (GraphPad Software Inc., San Diego,Calif.) and Excel program (Microsoft Corporation, Redmond, Wash.).

Using the assays above, compounds of the present invention aredetermined to be useful serine protease inhibitors.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of inhibiting the activity of a serineprotease comprising the step of contacting said serine protease with acompound of formula (I)

or a pharmaceutically acceptable salt thereof wherein: Each A is—(CX₁X₂)_(a)—; Each B is —(CX₁X₂)_(b)—; Each X₁ is independentlyhydrogen, halo, amino, sulfanyl, optionally substituted(C₁₋₄)-aliphatic, optionally substituted aryl, or —O—X_(1A); Each X₂ isindependently hydrogen, halo, amino, sulfanyl, optionally substituted(C₁₋₄)-aliphatic, optionally substituted aryl, or —O—X_(1B); X_(1A) andX_(1B) are each independently an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl; Or, X₁ and X₂ together form an oxo group; EachR₁ is

wherein T is a bond, —C(O)—, —OC(O)—, —NHC(O)—, —S(O)₂N(H)—, —C(O)C(O)—or —SO₂—; each R is independently hydrogen, amino, an optionallysubstituted aliphatic, an optionally substituted cycloaliphatic, anoptionally substituted heterocycloaliphatic, an optionally substitutedaryl, or an optionally substituted heteroaryl; each R₈ and R′₈ isindependently hydrogen, an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl; and each R₉ is independently hydrogen, anoptionally substituted aliphatic, an optionally substituted heteroaryl,an optionally substituted phenyl, or R₈ and R₉, bound on adjacent atoms,taken together with the atoms to which they are attached form a 5 to 7membered, optionally substituted monocyclic heterocycloaliphatic, or a 6to 12 membered, optionally substituted bicyclic heterocycloaliphatic; orR₈ and R′₈, taken together with the atoms to which they are attachedform an optionally substituted cycloaliphatic or an optionallysubstituted heterocycloaliphatic; Each R₂ is —Z^(B)R₅, wherein eachZ^(B) is independently a bond or an optionally substituted branched orstraight C₁₋₁₂ aliphatic chain wherein up to three carbon units of Z^(B)are optionally and independently replaced by —C(O)—, —C(S)—,—C(O)NR^(B)—, —C(O)NR^(B)NR^(B)—, —C(O)O—, —NR^(B)C(O)O—,—NR^(B)C(O)NR^(B)—, —NR^(B)NR^(B)—, —S—, —SO—, —SO₂—, —NR^(B)—,—SO₂NR^(B)—, or —NR^(B)SO₂NR^(B)—, provided that SO, SO₂, or —SO₂NR^(B)—is not directly bound to the carbonyl of formula I; Each R₅ isindependently R^(B), halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃; Each R^(B) isindependently hydrogen, an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substituted aryl,or an optionally substituted heteroaryl; Or R₁ and R₂, together with theatoms to which they are attached, form an optionally substitutedheterocycloaliphatic ring; Each R₃ is an optionally substitutedaliphatic, amino, sulfonyl, sulfanyl, sulfinyl, sulfonamide, sulfamide,sulfo, —O—R_(3A), an optionally substituted cycloaliphatic, anoptionally substituted heterocycloaliphatic, an optionally substitutedaryl, or an optionally substituted heteroaryl; Each R_(3A) isindependently an optionally substituted aliphatic, an optionallysubstituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl; Each Y and Y′ is independently —Z^(D)R₇, whereineach Z^(D) is independently a bond or an optionally substituted straightor branched C₁₋₆ aliphatic chain wherein up to two carbon units of Z^(D)are optionally and independently replaced by —C(O)—, —C(S)—,—C(O)NR^(D)—, —C(O)NR^(D)NR^(D)—, —C(O)O—, —NR^(D)C(O)O—, —O—,—NR^(D)C(O)NR^(D)—, —NR^(D)NR^(D)—, —S—, —SO—, —SO₂—, —NR^(D)—,—SO₂NR^(D)—, —NR^(D)SO₂—, or —NR^(D)SO₂NR^(D)—, or Y and Y′ togetherform ═O or ═S; Each R₇ is independently R^(D), halo, —OH, —CN, —NO₂,—NH₂, or —OCF₃; Each R^(D) is independently hydrogen, or optionallysubstituted aryl; and Each of a and b is independently 0, 1, 2, or 3;provided that the sum of a and b is 2 or
 3. 2. A method of inhibitingthe activity of a serine protease comprising the step of contacting saidserine protease with a compound selected from the group of compounds:


3. The method according to claim 1, wherein said serine protease is anHCV NS3 protease.
 4. The method according to claim 2, wherein saidserine protease is an HCV NS3 protease.
 5. A method of treating an HCVinfection in a patient comprising the step of administering to saidpatient a compound of formula (I)

or a pharmaceutically acceptable salt thereof wherein: Each A is—(CX₁X₂)_(a)—; Each B is —(CX₁X₂)_(b)—; Each X₁ is independentlyhydrogen, halo, amino, sulfanyl, optionally substituted(C₁₋₄)-aliphatic, optionally substituted aryl, or —O—X_(1A); Each X₂ isindependently hydrogen, halo, amino, sulfanyl, optionally substituted(C₁₋₄)-aliphatic, optionally substituted aryl, or —O—X_(1B); X_(1A) andX_(1B) are each independently an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl; Or, X₁ and X₂ together form an oxo group; EachR₁ is

wherein T is a bond, —C(O)—, —OC(O)—, —NHC(O)—, —S(O)₂N(H)—, —C(O)C(O)—or —SO₂—; each R is independently hydrogen, amino, an optionallysubstituted aliphatic, an optionally substituted cycloaliphatic, anoptionally substituted heterocycloaliphatic, an optionally substitutedaryl, or an optionally substituted heteroaryl; each R₈ and R′₈ isindependently hydrogen, an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl; and each R₉ is independently hydrogen, anoptionally substituted aliphatic, an optionally substituted heteroaryl,an optionally substituted phenyl, or R₈ and R₉, bound on adjacent atoms,taken together with the atoms to which they are attached form a 5 to 7membered, optionally substituted monocyclic heterocycloaliphatic, or a 6to 12 membered, optionally substituted bicyclic heterocycloaliphatic; orR₈ and R′₈, taken together with the atoms to which they are attachedform an optionally substituted cycloaliphatic or an optionallysubstituted heterocycloaliphatic; Each R₂ is —Z^(B)R₅, wherein eachZ^(B) is independently a bond or an optionally substituted branched orstraight C₁₋₁₂ aliphatic chain wherein up to three carbon units of Z^(B)are optionally and independently replaced by —C(O)—, —C(S)—,—C(O)NR^(B)—, —C(O)NR^(B)NR^(B)—, —C(O)O—, —NR^(B)C(O)O—,—NR^(B)C(O)NR^(B)—, —NR^(B)NR^(B)—, —S—, —SO—, —SO₂—, —NR^(B)—,—SO₂NR^(B)—, or —NR^(B)SO₂NR^(B)—, provided that SO, SO₂, or —SO₂NR^(B)—is not directly bound to the carbonyl of formula I; Each R₅ isindependently R^(B), halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃; Each R^(B) isindependently hydrogen, an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substituted aryl,or an optionally substituted heteroaryl; Or R₁ and R₂, together with theatoms to which they are attached, form an optionally substitutedheterocycloaliphatic ring; Each R₃ is an optionally substitutedaliphatic, amino, sulfonyl, sulfanyl, sulfinyl, sulfonamide, sulfamide,sulfa, —O—R_(3A), an optionally substituted cycloaliphatic, anoptionally substituted heterocycloaliphatic, an optionally substitutedaryl, or an optionally substituted heteroaryl; Each R_(3A) isindependently an optionally substituted aliphatic, an optionallysubstituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl; Each Y and Y′ is independently —Z^(D)R₇, whereineach Z^(D) is independently a bond or an optionally substituted straightor branched C₁₋₆ aliphatic chain wherein up to two carbon units of Z^(D)are optionally and independently replaced by —C(O)—, —C(S)—,—C(O)NR^(D)—, —C(O)NR^(D)NR^(D)—, —C(O)O—, —NR^(D)C(O)O—, —O—,—NR^(D)C(O)NR^(D)—, —NR^(D)NR^(D)—, —S—, —SO—, —SO₂—, —NR^(D)—,—SO₂NR^(D)—, —NR^(D)SO₂—, or —NR^(D)SO₂NR^(D)—, or Y and Y′ togetherform ═O or ═S; Each R₇ is independently R^(D), halo, —OH, —CN, —NO₂,—NH₂, or —OCF₃; Each R^(D) is independently hydrogen, or optionallysubstituted aryl; and Each of a and b is independently 0, 1, 2, or 3;provided that the sum of a and b is 2 or
 3. 6. A method of treating anHCV infection in a patient comprising the step of administering to saidpatient a compound selected from the group of compounds:


7. The method according to claim 5, further comprising administering tosaid patient an agent selected from an immunomodulatory agent; anantiviral agent; an inhibitor of HCV protease; an inhibitor of anothertarget in the HCV life cycle; or combinations thereof; wherein saidagent is administered to said patient in the same dosage form as theserine protease inhibitor or as a separate dosage form.
 8. The methodaccording to claim 6, further comprising administering to said patientan agent selected from an immunomodulatory agent; an antiviral agent; aninhibitor of HCV protease; an inhibitor of another target in the HCVlife cycle; or combinations thereof; wherein said agent is administeredto said patient in the same dosage form as the serine protease inhibitoror as a separate dosage form.
 9. The method according to claim 7,wherein said immunomodulatory agent is α-, β-, or γ-interferon orthymosin; said antiviral agent is ribavarin or amantadine; or saidinhibitor of another target in the HCV life cycle is an inhibitor of HCVhelicase, polymerase, or metalloprotease.
 10. The method according toclaim 8, wherein said immunomodulatory agent is α-, β-, or γ-interferonor thymosin; said antiviral agent is ribavarin or amantadine; or saidinhibitor of another target in the HCV life cycle is an inhibitor of HCVhelicase, polymerase, or metalloprotease.
 11. A method of eliminating orreducing HCV contamination of a biological sample or medical orlaboratory equipment, comprising the step of contacting said biologicalsample or medical or laboratory equipment with a compound of formula (I)

or a pharmaceutically acceptable salt thereof wherein: Each A is—(CX₁X₂)_(a)—; Each B is —(CX₁X₂)_(b)—; Each X₁ is independentlyhydrogen, halo, amino, sulfanyl, optionally substituted(C₁₋₄)-aliphatic, optionally substituted aryl, or —O—X_(1A); Each X₂ isindependently hydrogen, halo, amino, sulfanyl, optionally substituted(C₁₋₄)-aliphatic, optionally substituted aryl, or —O—X_(1B); X_(1A) and_(X) _(1B) are each independently an optionally substituted aliphatic,an optionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl; Or, X₁ and X₂ together form an oxo group; EachR₁ is

wherein T is a bond, —C(O)—, —OC(O)—, —NHC(O)—, —S(O)₂N(H)—, —C(O)C(O)—or —SO₂—; each R is independently hydrogen, amino, an optionallysubstituted aliphatic, an optionally substituted cycloaliphatic, anoptionally substituted heterocycloaliphatic, an optionally substitutedaryl, or an optionally substituted heteroaryl; each R₈ and R′₈ isindependently hydrogen, an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl; and each R₉ is independently hydrogen, anoptionally substituted aliphatic, an optionally substituted heteroaryl,an optionally substituted phenyl, or R₈ and R₉, bound on adjacent atoms,taken together with the atoms to which they are attached form a 5 to 7membered, optionally substituted monocyclic heterocycloaliphatic, or a 6to 12 membered, optionally substituted bicyclic heterocycloaliphatic; orR₈ and R′₈, taken together with the atoms to which they are attachedform an optionally substituted cycloaliphatic or an optionallysubstituted heterocycloaliphatic; Each R₂ is —Z^(B)R₅, wherein eachZ^(B) is independently a bond or an optionally substituted branched orstraight C₁₋₁₂ aliphatic chain wherein up to three carbon units of Z^(B)are optionally and independently replaced by —C(O)—, —C(S)—,—C(O)NR^(B)—, —C(O)NR^(B)NR^(B)—, —C(O)O—, —NR^(B)C(O)O—,—NR^(B)C(O)NR^(B)—, —NR^(B)NR^(B)—, —S—, —SO—, —SO₂—, —NR^(B)—,—SO₂NR^(B)—, or —NR^(B)SO₂NR^(B)—, provided that SO, SO₂, or —SO₂NR^(B)—is not directly bound to the carbonyl of formula I; Each R₅ isindependently R^(B), halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃; Each R^(B) isindependently hydrogen, an optionally substituted aliphatic, anoptionally substituted cycloaliphatic, an optionally substituted aryl,or an optionally substituted heteroaryl; Or R₁ and R₂, together with theatoms to which they are attached, form an optionally substitutedheterocycloaliphatic ring; Each R₃ is an optionally substitutedaliphatic, amino, sulfonyl, sulfanyl, sulfinyl, sulfonamide, sulfamide,sulfo, —O—R_(3A), an optionally substituted cycloaliphatic, anoptionally substituted heterocycloaliphatic, an optionally substitutedaryl, or an optionally substituted heteroaryl; Each R_(3A) isindependently an optionally substituted aliphatic, an optionallysubstituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl; Each Y and Y′ is independently —Z^(D)R₇, whereineach Z^(D) is independently a bond or an optionally substituted straightor branched C₁₋₆ aliphatic chain wherein up to two carbon units of Z^(D)are optionally and independently replaced by —C(O)—, —C(S)—,—C(O)NR^(D)—, —C(O)NR^(D)NR^(D)—, —C(O)O—, —NR^(D)C(O)O—, —O—,—NR^(D)C(O)NR^(D)—, —NR^(D)NR^(D)—, —S—, —SO—, —SO₂—, —NR^(D)—,—SO₂NR^(D)—, —NR^(D)SO₂—, or —NR^(D)SO₂NR^(D)—, or Y and Y′ togetherform ═O or ═S; Each R₇ is independently R^(D), halo, —OH, —CN, —NO₂,—NH₂, or —OCF₃; Each R^(D) is independently hydrogen, or optionallysubstituted aryl; and Each of a and b is independently 0, 1, 2, or 3;provided that the sum of a and b is 2 or
 3. 12. A method of eliminatingor reducing HCV contamination of a biological sample or medical orlaboratory equipment, comprising the step of contacting said biologicalsample or medical or laboratory equipment with a compound selected fromthe group of compounds: